Team:DTU Denmark/protocols



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   Table 2: Standard PCR program with Phusion polymerase. * for primers &gt; 20nt, anneal for 10 – 30 seconds at a Tm +3°C of the lower Tm primer. for primers ≤ 20nt, use an annealing temperature equal to the Tm of the lower Tm primer for calculating of melting temperature see Primer design</SPAN>. **15 sec per kb for low complexity DNA (e.g. plasmid, lambda or BAC DNA). 30 sec per kb for high complexity genomic DNA</FONT></P> <A name=_Toc243890358></A><A name=_Toc235693635></A><A name=_Toc235602392></A><A name=_Toc219878377></A><A name=_Toc219522883><SPAN style="mso-bookmark: _Toc219878377"><SPAN style="mso-bookmark: _Toc235602392"><SPAN style="mso-bookmark: _Toc235693635"><SPAN style="mso-bookmark: _Toc243890358"><FONT face=Calibri size=3>Taq polymerase</FONT></STRONG></SPAN></SPAN></SPAN></SPAN></A></P> <DIV align=center> </DIV> <B style="mso-bidi-font-weight: normal"><FONT face=Calibri size=2> </FONT></o:p></B></P> <FONT size=2><FONT face=Calibri><B style="mso-bidi-font-weight: normal">Table </B><B style="mso-bidi-font-weight: normal"><SPAN style="mso-no-proof: yes">3</SPAN></B></FONT></FONT><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri>: Standard program with Taq Polymerase. <BR>*Annealing temperatures should be chosen to match the Tm values of the primer pair. <BR>**use <SPAN class=bodycopy>1 minute/kb</SPAN></o:p></FONT></FONT></B></P> <A name=_Toc243890359></A><A name=_Toc235693636></A><A name=_Toc235602393></A><A name=_Toc219878378></A><A name=_Toc219522885></A><A name=_Toc188804807><SPAN style="mso-bookmark: _Toc219522885"><SPAN style="mso-bookmark: _Toc219878378"><SPAN style="mso-bookmark: _Toc235602393"><SPAN style="mso-bookmark: _Toc235693636"><SPAN style="mso-bookmark: _Toc243890359"><FONT face=Calibri size=3>Colony PCR</FONT></STRONG></SPAN></SPAN></SPAN></SPAN></SPAN></A></P> <FONT face=Calibri size=2>For each colonyPCR to be performed a PCR tube is filled with 35 µl MiliQ Water. A sterile tooth pick which has touched a desired colony is stirred around in the water for the bacteria to stay in the water. Then primers, buffer, dNTP and DNA Polymerase (usually Taq) is added in the same amounts as stated earlier for PCR reactions</FONT></P> <FONT size=2><FONT face=Calibri>A similar program as the before mentioned standard is used, with the modification of starting with 3 minutes at <SPAN style="COLOR: black">98º C to get the bacteria to lyse so the DNA is available for the primers to anneal.</o:p></SPAN></FONT></FONT></P> <DIV align=center> </DIV> <FONT face=Calibri size=2> </STRONG></FONT></o:p></P> <FONT face=Calibri size=2>Table <SPAN style="mso-no-proof: yes">4</SPAN>: Typical colony PCR program. </STRONG></FONT></P> <P class=Niveau3HC style="MARGIN: 0cm 0cm 0pt"><A name=_Toc243890360></A><A name=_Toc235693637></A><A name=_Toc235602394></A><A name=_Toc219878379></A><A name=_Toc219522886></A><A name=_Ref219110329><SPAN style="mso-bookmark: _Toc219522886"><SPAN style="mso-bookmark: _Toc219878379"><SPAN style="mso-bookmark: _Toc235602394"><SPAN style="mso-bookmark: _Toc235693637"><SPAN style="mso-bookmark: _Toc243890360"><STRONG><FONT face=Calibri size=3>Primer design</FONT></STRONG></SPAN></SPAN></SPAN></SPAN></SPAN></A></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>The annealing temperature (T<SUB>m</SUB>) of a primer is an important value to set in a PCR reaction is by definition the temperature at which one half of the DNA duplex will dissociate to become single stranded. At temperatures lower than the melting temperatures unspecific binding is possible so best PCR results is achieved at an annealing temperature close to the lowest melting temperature of the two primers. This means that primer pairs that have similar melting temperatures will yield a PCR reaction with reduced background noise by unspecific binding of the primers to the template.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>T<SUB>m</SUB> can be calculated using the “Oligo analyzer” </FONT><A href="http://www.genelink.com/tools/gl-downloads.asp"><U><FONT face=Calibri color=#0000ff size=2>www.genelink.com/tools/gl-downloads.asp</FONT></U></A></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Or be approximated using: <I>T<SUB>m</SUB></I>(°C) <SPAN style="mso-fareast-font-family: SymbolMT">≈ </SPAN>2(<I>N<SUB>A</SUB>+N<SUB>T</SUB></I>) <SPAN style="mso-fareast-font-family: SymbolMT">+ </SPAN>4(<I>N<SUB>G</SUB>+N<SUB>C</SUB></I>)</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>The primers described in this report is designed using </FONT><A href="http://fokker.wi.mit.edu/primer3/input.htm"><U><FONT face=Calibri color=#0000ff size=2>http://fokker.wi.mit.edu/primer3/input.htm</FONT></U></A><FONT face=Calibri size=2>, which is a handy tool to find primer pairs to amplify a specific sequence and discards potential primers<SPAN style="mso-spacerun: yes"> </SPAN>that have a tendency to e.g. self anneal. Usually a primer length of 22 was used based on experience. Primers was ordered at </FONT><A href="http://www.sigma.com/"><U><FONT face=Calibri color=#0000ff size=2>www.sigma.com</FONT></U></A><FONT face=Calibri size=2>.</FONT></P> <H2 style="MARGIN: 12pt 0cm 4pt"><A name=_Toc219522888></A><A name=_Toc243890361></A><A name=_Toc235693638></A><A name=_Toc235602395></A><A name=_Toc220487562></A><A name=_Toc219878380></A><A name=_Toc219522895></A><A name=_Ref219522840><SPAN style="mso-bookmark: _Toc219522895"><SPAN style="mso-bookmark: _Toc219878380"><SPAN style="mso-bookmark: _Toc220487562"><SPAN style="mso-bookmark: _Toc235602395"><SPAN style="mso-bookmark: _Toc235693638"><SPAN style="mso-bookmark: _Toc243890361"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=5>DNA sequencing</FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></A><SPAN style="mso-bookmark: _Toc219522888"></SPAN></H2> <P class=Niveau3HC style="MARGIN: 0cm 0cm 0pt"><SPAN style="mso-bookmark: _Toc219522888"><A name=_Toc243890362></A><A name=_Toc235693639></A><A name=_Toc235602396></A><A name=_Toc219878381></A><A name=_Toc219522896><SPAN style="mso-bookmark: _Toc219878381"><SPAN style="mso-bookmark: _Toc235602396"><SPAN style="mso-bookmark: _Toc235693639"><SPAN style="mso-bookmark: _Toc243890362"><FONT face=Calibri size=3>Preparing and sending DNA</FONT></SPAN></SPAN></SPAN></SPAN></A></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2>Description from </FONT></SPAN><A href="http://www.starseq.com/nomenu.php?nomenu=1&amp;ln=7n8"><U><FONT color=#0000ff><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522888">http://www.starseq.com/nomenu.php?nomenu=1&amp;ln=7n8</SPAN><SPAN style="mso-bookmark: _Toc219522888"></SPAN></FONT></FONT></FONT></U></A><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2> </FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT size=2><FONT face=Calibri><I style="mso-bidi-font-style: normal">Mix </I>Each PCR tube contains DNA, MQ H<SUB>2</SUB>O and a single sequence primer in a total volume of 6μl. Add 3-5 μl plasmid and 1μl primer – see <SPAN style="mso-field-code: ' REF _Ref219107281 h * MERGEFORMAT '">Table <SPAN style="mso-no-proof: yes">5</SPAN></SPAN>. </FONT></FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT size=2><FONT face=Calibri><I style="mso-bidi-font-style: normal">Label</I><B style="mso-bidi-font-weight: normal"> </B>legible, with indelible black pen (e. g. Staedtler permanent lumocolor; Art. Nr. 318-9; EAN 40 07817 304563 or similar). Note your abbreviation on the lids and number them continuously.</FONT></FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT size=2><FONT face=Calibri><I style="mso-bidi-font-style: normal">Send</I><B style="mso-bidi-font-weight: normal"> </B>Protect the reaction containers with a box or something similar and send together with order form in a padded envelope to: StarSEQ, GENterprise GMBH, Johann-Joachim-Becher-Weg 30a, 55099 Mainz, Germany´</FONT></FONT></SPAN></P> <P class=MsoCaption style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><A name=_Ref219107281><STRONG><FONT face=Calibri size=2>Table </FONT></STRONG></A></SPAN><STRONG><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="mso-bookmark: _Ref219107281"><SPAN style="mso-no-proof: yes">5</SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="mso-bookmark: _Ref219107281"></SPAN>: Relevant sequencing informations</SPAN></FONT></FONT></STRONG></P> <TABLE class=MsoNormalTable style="BORDER-RIGHT: medium none; BORDER-TOP: medium none; BORDER-LEFT: medium none; WIDTH: 451.95pt; BORDER-BOTTOM: medium none; BORDER-COLLAPSE: collapse; mso-yfti-tbllook: 480; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-border-top-alt: solid windowtext 1.5pt; mso-border-bottom-alt: solid windowtext 1.5pt" cellSpacing=0 cellPadding=0 width=603 border=1> <TBODY> <TR style="mso-yfti-irow: 0; mso-yfti-firstrow: yes"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: windowtext 1.5pt solid; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><STRONG><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">DNA</SPAN><o:p></o:p></FONT></FONT></STRONG></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: windowtext 1.5pt solid; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>dissolved in water or TrisHCl (10mM), pH 7-8</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 1"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Amounts</SPAN><o:p></o:p></FONT></FONT></B></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>PCR product: 200 bp: 50 ng, 500 bp: 100 ng; 1 kb: 200 ng</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 2"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Plasmid DNA</SPAN><o:p></o:p></FONT></FONT></B></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>400 ng - 700 ng</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 3"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Cosmid DNA, PACs, BACs</SPAN><o:p></o:p></FONT></FONT></B></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>&gt;1 µg</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 4"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Primer</SPAN><o:p></o:p></FONT></FONT></B></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>10 pmol, i.e. 1 µl of a 10 µMolar primer solution</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 5"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Melting temperature</SPAN><o:p></o:p></FONT></FONT></B></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>52 - 60°C</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 6"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Optimal length</SPAN><o:p></o:p></FONT></FONT></B></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>18 - 25mer</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 7; mso-yfti-lastrow: yes"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 161.35pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=215> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Tube</SPAN><o:p></o:p></FONT></FONT></B></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 290.6pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=387> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>200 µl PCR tubes with flat lids (e. g. Starlab, Art. Nr. I 1402-8100 or similar); Close only, no Parafilm</FONT></SPAN></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2></FONT></SPAN></TR></TBODY></TABLE> <P class=Niveau3HC style="MARGIN: 0cm 0cm 0pt"><SPAN style="mso-bookmark: _Toc219522888"><A name=_Toc243890363></A><A name=_Toc235693640></A><A name=_Toc235602397></A><A name=_Toc219878382></A><A name=_Toc219522897><SPAN style="mso-bookmark: _Toc219878382"><SPAN style="mso-bookmark: _Toc235602397"><SPAN style="mso-bookmark: _Toc235693640"><SPAN style="mso-bookmark: _Toc243890363"><STRONG><FONT face=Calibri size=3>Analyzing retrieved files</FONT></STRONG></SPAN></SPAN></SPAN></SPAN></A></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2>The files return as SEQ and AB1 files. AB1 files can be opened with the freeware program FinchTV.</FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2>Open all files. In each file the sequence that look reliable (normally from base 20 to ~700) is copied and pasted in a txt file. </FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2>Assemble the contigs using Vector NTI from Invitrogen</FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2>Use the “Assemble” function, and click“Open New Assembly Project”. </FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2>Add fragments (txt. files)</FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522888"><FONT face=Calibri size=2>Select fragments and assemble. </FONT></SPAN></P> <H2 style="MARGIN: 12pt 0cm 4pt"><SPAN style="mso-bookmark: _Toc219522888"><A name=_Toc243890364></A><A name=_Toc235693641></A><A name=_Toc235602398></A><A name=_Toc220487563></A><A name=_Toc219878383><SPAN style="mso-bookmark: _Toc220487563"><SPAN style="mso-bookmark: _Toc235602398"><SPAN style="mso-bookmark: _Toc235693641"><SPAN style="mso-bookmark: _Toc243890364"><FONT face=Calibri size=5>Digestion with restriction enzymes</FONT></SPAN></SPAN></SPAN></SPAN></A></SPAN></H2> <P class=Niveau3HC style="MARGIN: 0cm 0cm 0pt"><A name=_Toc243890365></A><A name=_Toc235693642></A><A name=_Toc235602399></A><A name=_Toc219878384></A><A name=_Toc219522889><SPAN style="mso-bookmark: _Toc219878384"><SPAN style="mso-bookmark: _Toc235602399"><SPAN style="mso-bookmark: _Toc235693642"><SPAN style="mso-bookmark: _Toc243890365"><FONT face=Calibri size=3>Materials and chemicals</FONT></SPAN></SPAN></SPAN></SPAN></A></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>MiliQ water</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Restriction enzymes 10,000 U/ml</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>NEBuffer 10X (1,2,3,4)</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Bovine serum albumin (BSA) 100X (10X)</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>DNA </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>All restriction enzymes, enzyme buffers and bovine serum albumin (BSA) used in this study were purchased through New England Biolabs. Information about specific enzyme conditions e.g. type of NEBuffer and whether BSA was needed or not was obtained through the NEB website: </FONT><A href="http://www.neb.com/nebecomm/products/category1.asp?#2"><U><FONT face=Calibri color=#0000ff size=2>http://www.neb.com/nebecomm/products/category1.asp?#2</FONT></U></A><FONT face=Calibri size=2>.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri>Initial concentrations of NEBuffers were provided as 10X and diluted to 1X in the final mix. BSA was provided as 10 mg/ml (100X) and should optimally have a final concentration of 100 μg/ml (1X). In this study the original BSA solution had been diluted to 10X which then was used as stock. Enzymes that do not require BSA should not be affected if BSA is present. <SPAN class=bodycopy><o:p></o:p></SPAN></FONT></FONT></P> <P class=Niveau3HC style="MARGIN: 0cm 0cm 0pt"><A name=_Toc243890366></A><A name=_Toc235693643></A><A name=_Toc235602400></A><A name=_Toc219878385></A><A name=_Toc219522890><SPAN style="mso-bookmark: _Toc219878385"><SPAN style="mso-bookmark: _Toc235602400"><SPAN style="mso-bookmark: _Toc235693643"><SPAN style="mso-bookmark: _Toc243890366"><SPAN class=bodycopy><STRONG><FONT face=Calibri size=3>Procedure</FONT></STRONG></SPAN></SPAN></SPAN></SPAN></SPAN></A><SPAN style="mso-bookmark: _Toc243890366"></SPAN><SPAN style="mso-bookmark: _Toc235693643"></SPAN><SPAN style="mso-bookmark: _Toc235602400"></SPAN><SPAN style="mso-bookmark: _Toc219878385"></SPAN><SPAN style="mso-bookmark: _Toc219522890"></SPAN><SPAN class=bodycopy><SPAN style="FONT-WEIGHT: normal; FONT-SIZE: 10pt; LINE-HEIGHT: 115%; LETTER-SPACING: 0pt; FONT-VARIANT: normal! important"><o:p></o:p></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><SPAN class=bodycopy><I style="mso-bidi-font-style: normal">Short protocol</I> MiliQ H<SUB>2</SUB>O was added first, buffer next, BSA when required, then the DNA solution, and finally the enzyme. The reaction mixture was mixed by gently pipetting up and down or by flicking the tube. </SPAN><SPAN class=bodycopy><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 115%; LETTER-SPACING: 0.25pt; FONT-VARIANT: small-caps"><o:p></o:p></SPAN></B></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN class=bodycopy><FONT size=2><FONT face=Calibri><I style="mso-bidi-font-style: normal">Amount of enzyme</I> Generally, 10 U (1 μl) enzyme is normally added to 1 μg of purified DNA in a final volume of 50 μl. Thus, the amount of enzyme depends on the DNA concentration which can be verified by gel electrophoresis. A little more enzyme was added when double digestion was performed with enzymes requiring different types of buffers for optimal activity. E.g. 30 U of AsiSI and 40 U of KpnI was used with NEBuffer 2, because KpnI has got only 75% activity in this buffer and AsiSI had 100%. <o:p></o:p></FONT></FONT></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><SPAN class=bodycopy><I style="mso-bidi-font-style: normal">Incubation </I></SPAN>In the 1st hour of incubation most of the DNA will be digested according to NEB, but for most of our reactions a more complete digestion was obtained by incubating “over night”. When this was done new enzymes were added halfway. All reaction were incubated overnight at 37º C. </FONT></FONT></P> <TABLE class=MsoNormalTable style="BORDER-RIGHT: medium none; BORDER-TOP: medium none; BORDER-LEFT: medium none; BORDER-BOTTOM: medium none; BORDER-COLLAPSE: collapse; mso-yfti-tbllook: 480; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-border-top-alt: solid windowtext 1.5pt; mso-border-insideh: 1.5pt solid windowtext" cellSpacing=0 cellPadding=0 border=1> <TBODY> <TR style="mso-yfti-irow: 0; mso-yfti-firstrow: yes"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: windowtext 1.5pt solid; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 83.4pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=111> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Enzyme</SPAN><o:p></o:p></FONT></FONT></B></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: windowtext 1.5pt solid; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 148.8pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=198> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Recognition site</SPAN><o:p></o:p></FONT></FONT></B></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: windowtext 1.5pt solid; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 219.75pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=293> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><B style="mso-bidi-font-weight: normal"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Used for</SPAN><o:p></o:p></FONT></FONT></B></P></TD></TR> <TR style="mso-yfti-irow: 1"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 83.4pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext 1.5pt" width=111> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><B style="mso-bidi-font-weight: normal"><I style="mso-bidi-font-style: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Kpn</SPAN></I></B><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">I</SPAN><o:p></o:p></B></FONT></FONT></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 148.8pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext 1.5pt" width=198> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-language: AR-SA; mso-bidi-font-size: 11.0pt; mso-no-proof: yes"><v:shape id=Billede_x0020_3 style="VISIBILITY: visible; WIDTH: 98.25pt; HEIGHT: 24pt; mso-wrap-style: square" alt="GGTACC" type="#_x0000_t75" o:spid="_x0000_i1030"><v:imagedata o:title="GGTACC" src="file:///C:\DOCUME~1\Mads\LOCALS~1\Temp\msohtmlclip1\01\clip_image003.png"><FONT face=Calibri size=2></FONT></v:imagedata></v:shape></SPAN><o:p></o:p></B></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 219.75pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext 1.5pt" width=293> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>Cloning </FONT></SPAN></P></TD></TR> <TR style="mso-yfti-irow: 2"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 83.4pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=111> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><B style="mso-bidi-font-weight: normal"><I style="mso-bidi-font-style: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Asi</SPAN></I></B><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">SI</SPAN><o:p></o:p></B></FONT></FONT></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 148.8pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=198> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-language: AR-SA; mso-bidi-font-size: 11.0pt; mso-no-proof: yes"><v:shape id=Billede_x0020_4 style="VISIBILITY: visible; WIDTH: 104.25pt; HEIGHT: 24pt; mso-wrap-style: square" type="#_x0000_t75" o:spid="_x0000_i1029"><v:imagedata o:title="" src="file:///C:\DOCUME~1\Mads\LOCALS~1\Temp\msohtmlclip1\01\clip_image004.png"><FONT face=Calibri size=2></FONT></v:imagedata></v:shape></SPAN><o:p></o:p></B></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 219.75pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=293> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt"><FONT face=Calibri size=2>Cloning, Restriction Analysis</FONT></SPAN></P></TD></TR> <TR style="mso-yfti-irow: 3"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 83.4pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=111> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><B style="mso-bidi-font-weight: normal"><I style="mso-bidi-font-style: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Apa</SPAN></I></B><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">LI</SPAN><o:p></o:p></B></FONT></FONT></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 148.8pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=198> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-language: AR-SA; mso-bidi-font-size: 11.0pt; mso-no-proof: yes"><v:shape id=Billede_x0020_5 style="VISIBILITY: visible; WIDTH: 96pt; HEIGHT: 24pt; mso-wrap-style: square" alt="GTGCAC" type="#_x0000_t75" o:spid="_x0000_i1028"><v:imagedata o:title="GTGCAC" src="file:///C:\DOCUME~1\Mads\LOCALS~1\Temp\msohtmlclip1\01\clip_image005.png"><FONT face=Calibri size=2></FONT></v:imagedata></v:shape></SPAN><o:p></o:p></B></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 219.75pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" width=293> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Restriction Analysis, linearized BGHA P8 before transformation</SPAN><B style="mso-bidi-font-weight: normal"><o:p></o:p></B></FONT></FONT></P></TD></TR> <TR style="mso-yfti-irow: 4; mso-yfti-lastrow: yes"> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 83.4pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=111> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><B style="mso-bidi-font-weight: normal"><I style="mso-bidi-font-style: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Not</SPAN></I></B><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">I</SPAN><o:p></o:p></B></FONT></FONT></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 148.8pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=198> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><B style="mso-bidi-font-weight: normal"><SPAN style="LINE-HEIGHT: 115%; mso-bidi-language: AR-SA; mso-bidi-font-size: 11.0pt; mso-no-proof: yes"><v:shape id=Billede_x0020_6 style="VISIBILITY: visible; WIDTH: 112.5pt; HEIGHT: 24pt; mso-wrap-style: square" alt="GCGGCCGC" type="#_x0000_t75" o:spid="_x0000_i1027"><v:imagedata o:title="GCGGCCGC" src="file:///C:\DOCUME~1\Mads\LOCALS~1\Temp\msohtmlclip1\01\clip_image006.png"><FONT face=Calibri size=2></FONT></v:imagedata></v:shape></SPAN><o:p></o:p></B></P></TD> <TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 1.4pt; BORDER-LEFT: #d4d0c8; WIDTH: 219.75pt; PADDING-TOP: 1.4pt; BORDER-BOTTOM: windowtext 1.5pt solid; BACKGROUND-COLOR: transparent" width=293> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><SPAN style="LINE-HEIGHT: 115%; mso-bidi-font-size: 11.0pt">Restriction Analysis</SPAN><B style="mso-bidi-font-weight: normal"><o:p></o:p></B></FONT></FONT></P></TD></TR></TBODY></TABLE> <P class=MsoCaption style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2><STRONG>Table <SPAN style="mso-no-proof: yes">6</SPAN>: Restriction endonucelases used in this study. </STRONG></FONT></P> <P class=Niveau3HC style="MARGIN: 0cm 0cm 0pt"><A name=_Toc243890367></A><A name=_Toc235693644></A><A name=_Toc235602401></A><A name=_Toc219878386></A><A name=_Toc219522891><SPAN style="mso-bookmark: _Toc219878386"><SPAN style="mso-bookmark: _Toc235602401"><SPAN style="mso-bookmark: _Toc235693644"><SPAN style="mso-bookmark: _Toc243890367"><STRONG><FONT face=Calibri size=3>More information</FONT></STRONG></SPAN></SPAN></SPAN></SPAN></A></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>NEB restriction enzymes:</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><A href="http://www.neb.com/nebecomm/products/faqCategory1.asp#661"><U><FONT face=Calibri color=#0000ff size=2>http://www.neb.com/nebecomm/products/faqCategory1.asp#661</FONT></U></A></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><o:p><FONT face=Calibri size=2> </FONT></o:p></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>NEBuffers:</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><A href="http://www.neb.com/nebecomm/products/productB7000.asp"><U><FONT face=Calibri color=#0000ff size=2>http://www.neb.com/nebecomm/products/productB7000.asp</FONT></U></A></P> <H2 style="MARGIN: 12pt 0cm 4pt"><A name=_Toc219522892></A><A name=_Toc243890368></A><A name=_Toc235693645></A><A name=_Toc235602402></A><A name=_Toc220487564></A><A name=_Toc219878387></A><A name=_Ref219526472></A><A name=_Toc219522910><SPAN style="mso-bookmark: _Ref219526472"><SPAN style="mso-bookmark: _Toc219878387"><SPAN style="mso-bookmark: _Toc220487564"><SPAN style="mso-bookmark: _Toc235602402"><SPAN style="mso-bookmark: _Toc235693645"><SPAN style="mso-bookmark: _Toc243890368"><SPAN style="mso-bookmark: _Toc219522892"><SPAN style="mso-no-proof: yes"><FONT face=Calibri size=5>Ligation</FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></A><SPAN style="mso-bookmark: _Toc219522892"></SPAN></H2> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><o:p><FONT face=Calibri size=2> </FONT></o:p></SPAN></P> <DIV align=center> <TABLE class=MsoNormalTable style="BORDER-RIGHT: medium none; BORDER-TOP: medium none; MARGIN: auto auto auto 23.4pt; BORDER-LEFT: medium none; BORDER-BOTTOM: medium none; BORDER-COLLAPSE: collapse; mso-yfti-tbllook: 191; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-border-insideh: .5pt solid windowtext; mso-border-alt: solid windowtext .5pt; mso-border-insidev: .5pt solid windowtext" cellSpacing=0 cellPadding=0 border=1> <TBODY> <TR style="mso-yfti-irow: 0; mso-yfti-firstrow: yes"> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: windowtext 1pt solid; WIDTH: 123.75pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-alt: solid windowtext .5pt" vAlign=top width=165> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2>1 µl ligase</FONT></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: windowtext 1pt solid; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: #d4d0c8; WIDTH: 127.6pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt" vAlign=top width=170><SPAN style="mso-bookmark: _Toc219522892"></SPAN> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><o:p><FONT face=Calibri size=2> </FONT></o:p></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 1"> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: windowtext 1pt solid; WIDTH: 123.75pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt" vAlign=top width=165> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2>4 µl 10x ligase buffer</FONT></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: #d4d0c8; WIDTH: 127.6pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt" vAlign=top width=170><SPAN style="mso-bookmark: _Toc219522892"></SPAN> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><o:p><FONT face=Calibri size=2> </FONT></o:p></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 2"> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: windowtext 1pt solid; WIDTH: 123.75pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt" vAlign=top width=165> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2>X µl plasmid, cut</FONT></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: #d4d0c8; WIDTH: 127.6pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt" vAlign=top width=170> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2>X = 50-100 ng plasmid</FONT></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 3"> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: windowtext 1pt solid; WIDTH: 123.75pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt" vAlign=top width=165> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2>Y µl PCR product, cut</FONT></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: #d4d0c8; WIDTH: 127.6pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt" vAlign=top width=170> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2>Y = 300 ng PCR product</FONT></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN></TR> <TR style="mso-yfti-irow: 4; mso-yfti-lastrow: yes"> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: windowtext 1pt solid; WIDTH: 123.75pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt" vAlign=top width=165> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2>Z µl MQ up to 40 µl total</FONT></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN> <TD style="BORDER-RIGHT: windowtext 1pt solid; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0cm; BORDER-LEFT: #d4d0c8; WIDTH: 127.6pt; PADDING-TOP: 0cm; BORDER-BOTTOM: windowtext 1pt solid; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid windowtext .5pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt" vAlign=top width=170><SPAN style="mso-bookmark: _Toc219522892"></SPAN> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><o:p><FONT face=Calibri size=2> </FONT></o:p></SPAN></P></TD><SPAN style="mso-bookmark: _Toc219522892"><FONT face=Calibri size=2></FONT></SPAN></TR></TBODY></TABLE></DIV> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><o:p><FONT face=Calibri size=2> </FONT></o:p></SPAN></P> <P class=MsoBodyText style="MARGIN: 0cm 0cm 12pt; TEXT-INDENT: 0cm; LINE-HEIGHT: normal"><FONT size=2><SPAN style="mso-bookmark: _Toc219522892"><SPAN style="FONT-FAMILY: 'Times New Roman','serif'; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi">Everything is mixed, and incubated – either at room temperature for 2 hours, or overnight using the idea of Lund <I style="mso-bidi-font-style: normal">et al</I> </SPAN></SPAN><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522892"><I style="mso-bidi-font-style: normal"><SPAN style="mso-no-proof: yes">(26)</SPAN></I></SPAN><SPAN style="mso-bookmark: _Toc219522892"></SPAN></FONT><SPAN style="mso-bookmark: _Toc219522892"><SPAN style="FONT-FAMILY: 'Times New Roman','serif'; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi"> of incubating on PCR machine with the following PCR program<o:p></o:p></SPAN></SPAN></FONT></P> <P class=MsoBodyText style="MARGIN: 0cm 0cm 12pt; TEXT-INDENT: 0cm; LINE-HEIGHT: normal"><SPAN style="mso-bookmark: _Toc219522892"><SPAN style="FONT-FAMILY: 'Times New Roman','serif'; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi"><o:p><FONT size=2> </FONT></o:p></SPAN></SPAN></P> <P class=MsoBodyText style="MARGIN: 0cm 0cm 12pt; TEXT-INDENT: 0cm; LINE-HEIGHT: normal"><SPAN style="mso-bookmark: _Toc219522892"><SPAN style="FONT-FAMILY: 'Times New Roman','serif'; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi"><o:p><FONT size=2> </FONT></o:p></SPAN></SPAN></P> <DIV align=center> </DIV> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><B style="mso-bidi-font-weight: normal"><o:p><FONT face=Calibri size=2> </FONT></o:p></B></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522892"><B style="mso-bidi-font-weight: normal">Table </B></SPAN><SPAN style="mso-bookmark: _Toc219522892"><B style="mso-bidi-font-weight: normal"><SPAN style="mso-no-proof: yes">7</SPAN></B></SPAN><SPAN style="mso-bookmark: _Toc219522892"><B style="mso-bidi-font-weight: normal">: <SPAN style="mso-bidi-font-weight: bold">Ligation program on PCR machine<o:p></o:p></SPAN></B></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522892"><SPAN style="COLOR: black"><BR></SPAN><FONT face=Calibri size=2>After the incubation, the mix can be stored at -18˚C. Lund et al </FONT></SPAN><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522892"><SPAN style="mso-no-proof: yes">(26)</SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522892"> showed that ligation with cohesive ends carried out on with the temperature shifts could give an increased ligation yield of 7.7 times. This is because ligase works best at <SPAN style="COLOR: black">30º C but template annealing is most effective at 10º C.</SPAN></SPAN></FONT></FONT></P><SPAN style="mso-bookmark: _Toc219522892"></SPAN> <H2 style="MARGIN: 12pt 0cm 4pt"><A name=_Toc243890369></A><A name=_Toc235693646></A><A name=_Toc235602403></A><A name=_Toc220487565></A><A name=_Toc219878388></A><A name=_Toc219522911><SPAN style="mso-bookmark: _Toc219878388"><SPAN style="mso-bookmark: _Toc220487565"><SPAN style="mso-bookmark: _Toc235602403"><SPAN style="mso-bookmark: _Toc235693646"><SPAN style="mso-bookmark: _Toc243890369"><FONT face=Calibri size=5>Miniprep for plasmid purification</FONT></SPAN></SPAN></SPAN></SPAN></SPAN></A><SPAN style="mso-bookmark: _Toc243890369"></SPAN><SPAN style="mso-bookmark: _Toc235693646"></SPAN><SPAN style="mso-bookmark: _Toc235602403"></SPAN><SPAN style="mso-bookmark: _Toc220487565"></SPAN><SPAN style="mso-bookmark: _Toc219878388"></SPAN><SPAN style="mso-bookmark: _Toc219522911"></SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 115%"><o:p></o:p></SPAN></H2> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>(Sigma GenElute<SUP>TM</SUP> Plasmid Miniprep Kit, from manual)</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l1 level1 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>1.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Harvest &amp; lyse bacteria</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>a.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">       </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Pellet cells from 1 – 5 ml overnight culture 1 min (1 ml from TB or 2xYT; 1-5 ml from LB medium). Discard supernatant.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>b.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Resuspend cells in 200 µl Resuspension Solution. Pipet up and down or vortex. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>c.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">       </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Add 200 µl of Lysis Solution. Invert gently to mix. Do not vortwx. Allow to clear for ≤ 5 min.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l1 level1 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>2.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Prepare cleared lysate</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>a.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">       </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Add 350 µl of Neutralization Solution (S3). Invert 4- 6 times to mix.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>b.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Pellet debris 10 min. at max speed.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l1 level1 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>3.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Prepare binding column</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>a.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">       </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Add 500 µl Column Preparation Solution to binding column in a collection tube.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>b.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Spin at ≥ 12.000 x g, 1 min. Discard flow-through.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l1 level1 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>4.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Bind plasmid DNA to column</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>a.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">       </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Transfer cleared lysate into binding column.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>b.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Spin 30” – 1 min. Discard flow-through.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l1 level1 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>5.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Wash to remove contaminants</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>a.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">       </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Optional (<I>EndA<SUP>+</SUP></I> <I>strains only</I>): Add 500 µl Optional Wash Solution to column. Spin 30” – 1 min. Discard flow-through.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>b.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Spin 1 min to dry column</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l1 level1 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>6.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Elute purified plasmid DNA</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>a.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">       </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Transfer column to new collection tube.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 54pt; TEXT-INDENT: -18pt; tab-stops: list 54.0pt; mso-list: l1 level2 lfo1"><SPAN style="mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin"><SPAN style="mso-list: Ignore"><FONT face=Calibri size=2>b.</FONT><SPAN style="FONT: 7pt 'Times New Roman'">      </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Add 100 µl Elution Solution. Spin 1min.</FONT></P> <P class=Style1 style="MARGIN: 12pt 0cm 4pt"><A name=_Toc219878392></A><A name=_Toc219522913></A><A name=_Toc243890370></A><A name=_Toc235693647></A><A name=_Toc235602404></A><A name=_Toc220487567></A><A name=_Toc219878391></A><A name=_Ref219541328><SPAN style="mso-bookmark: _Toc219878391"><SPAN style="mso-bookmark: _Toc220487567"><SPAN style="mso-bookmark: _Toc235602404"><SPAN style="mso-bookmark: _Toc235693647"><SPAN style="mso-bookmark: _Toc243890370"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><FONT face=Calibri size=5>DNA purification from gel</FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></A><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><FONT face=Calibri size=2>Using illustra<SUP>TM</SUP> GFX PCR DNA and Gel Band Purification Kit. Protocol from manual has been slightly altered by eluting using MiliQ Water and not elution buffer.</FONT></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><B><SPAN style="mso-fareast-font-family: Calibri"><FONT size=2><FONT face=Calibri>1. Sample Capture<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>a. Weigh a DNase-free 1.5 ml micro centrifuge tube and record the weight.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>b. Using a clean scalpel, long wavelength (365 nm) ultraviolet light and minimal exposure<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>time, cut out as small an agarose band as possible containing the sample of interest.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>Place agarose gel band into a DNase-free 1.5 ml microcentrifuge tube.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. Weigh the microcentrifuge tube plus agarose band and calculate the weight of the agarose<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>slice.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">d. Add 10 μl </SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: Calibri; mso-bidi-font-weight: bold">Capture buffer type 2<B> </B></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">for each 10 mg of gel slice.<o:p></o:p></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>e. Mix by inversion and incubate at 60°C until the agarose is completely dissolved. Mix by<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>inversion every 3 minutes.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>f. For each purification that is to be performed, place one GFX MicroSpin column into one Collection tube.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><B><SPAN style="mso-fareast-font-family: Calibri"><FONT size=2><FONT face=Calibri>2. Sample Binding<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">a. Centrifuge </SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: Calibri; mso-bidi-font-weight: bold">Capture buffer type 2</SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">- sample mix briefly to collect the liquid at the bottom of the tube. <o:p></o:p></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">b. Transfer 600 μl </SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: Calibri; mso-bidi-font-weight: bold">Capture buffer type 2</SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">- sample mix onto the assembled GFX MicroSpin column<o:p></o:p></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>and Collection tube.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. Incubate at room temperature for 1 minute.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>d. Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>e. Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>column back inside the Collection tube.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>f. Repeat Sample Binding steps b. to e. as necessary until all sample is loaded.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><B><SPAN style="mso-fareast-font-family: Calibri"><FONT size=2><FONT face=Calibri>3. Wash &amp; Dry<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">a. Add 500 μl </SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: Calibri; mso-bidi-font-weight: bold">Wash buffer type 1<B> </B></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">to the GFX MicroSpin column.<o:p></o:p></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>b. Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>microcentrifuge tube (supplied by user).<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><B><SPAN style="mso-fareast-font-family: Calibri"><FONT size=2><FONT face=Calibri>4. Elution<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">a. Add 10–50 μl Mq water</SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><B><SPAN style="mso-fareast-font-family: Calibri"> </SPAN></B></SPAN></SPAN><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira">to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.<o:p></o:p></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>b. Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. Spin the assembled column and sample Collection tube at 16 000 × g for 1 minute to recover the purified DNA.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><SPAN style="mso-fareast-font-family: GEInspira"><FONT face=Calibri size=2>d. Proceed to downstream application. Store the purified DNA at -20°C.</FONT></SPAN></SPAN></SPAN></P> <H2 style="MARGIN: 12pt 0cm 4pt"><SPAN style="mso-bookmark: _Toc219522913"><SPAN style="mso-bookmark: _Toc219878392"><A name=_Toc243890371></A><A name=_Toc235693648></A><A name=_Toc235602405></A><A name=_Toc220487568><SPAN style="mso-bookmark: _Toc235602405"><SPAN style="mso-bookmark: _Toc235693648"><SPAN style="mso-bookmark: _Toc243890371"><FONT face=Calibri size=5>DNA purification from enzymatic reaction</FONT></SPAN></SPAN></SPAN></A></SPAN></SPAN></H2> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Using illustra<SUP>TM</SUP> GFX PCR DNA and Gel Band Purification Kit. Protocol from manual has been slightly altered by eluting using MiliQ Water and not elution buffer.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><A name=_Toc219522916></A><A name=_Toc219878393></A><A name=_Toc219522887></A><A name=_Toc219522898></A><A name=_Ref219517590><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><B><SPAN style="mso-fareast-font-family: Calibri"><FONT size=2><FONT face=Calibri>1. Sample Capture<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></SPAN></SPAN></A></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira">a. Add 500 μl </SPAN></SPAN></SPAN></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: Calibri; mso-bidi-font-weight: bold">Capture buffer type 2<B> </B></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira">to up to 100 μl sample.<o:p></o:p></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>b. Mix thoroughly.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. For each purification that is to be performed, place one GFX MicroSpin column into one Collection tube.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><B><SPAN style="mso-fareast-font-family: Calibri"><FONT size=2><FONT face=Calibri>2. Sample Binding<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira">a. Centrifuge </SPAN></SPAN></SPAN></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: Calibri; mso-bidi-font-weight: bold">Capture buffer type 2</SPAN></SPAN></SPAN></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira">-sample mix briefly to collect the liquid at the bottom of the tube.<o:p></o:p></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><FONT size=2><FONT face=Calibri><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira">b. Load the </SPAN></SPAN></SPAN></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: Calibri; mso-bidi-font-weight: bold">Capture buffer type 2</SPAN></SPAN></SPAN></SPAN></SPAN></SPAN><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira">-sample mix onto the assembled GFX MicroSpin column and Collection tube.<o:p></o:p></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>d. Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin column back inside the Collection tube.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><B><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>3. Wash &amp; Dry<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>a. Add 500 μl <SPAN style="mso-bidi-font-weight: bold">Wash buffer type 1<B> </B></SPAN>to the GFX MicroSpin column.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>b. Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube (supplied by user).<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><B><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>4. Elution<o:p></o:p></FONT></FONT></SPAN></B></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>a. Add 10–50 μl Mq-water<B> </B>to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>b. Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT size=2><FONT face=Calibri>c. Spin the assembled column and sample Collection tube at 16 000 × g for 1 minute to recover the purified DNA.<o:p></o:p></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt; mso-layout-grid-align: none"><SPAN style="mso-bookmark: _Ref219517590"><SPAN style="mso-bookmark: _Toc219522898"><SPAN style="mso-bookmark: _Toc219522887"><SPAN style="mso-bookmark: _Toc219878393"><SPAN style="mso-bookmark: _Toc219522916"><SPAN style="mso-fareast-font-family: GEInspira"><FONT face=Calibri size=2>d. Proceed to downstream application. Store the purified DNA at -20°C.</FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></P><SPAN style="mso-bookmark: _Toc219878393"></SPAN><SPAN style="mso-bookmark: _Toc219522887"></SPAN><SPAN style="mso-bookmark: _Toc219522898"></SPAN><SPAN style="mso-bookmark: _Ref219517590"></SPAN> <H2 style="MARGIN: 12pt 0cm 4pt"><SPAN style="mso-bookmark: _Toc219522916"><A name=_Toc243890372></A><A name=_Toc235693649></A><A name=_Toc235602406></A><A name=_Toc220487573></A><A name=_Toc219878405></A><A name=_Ref219527486><SPAN style="mso-bookmark: _Toc219878405"><SPAN style="mso-bookmark: _Toc220487573"><SPAN style="mso-bookmark: _Toc235602406"><SPAN style="mso-bookmark: _Toc235693649"><SPAN style="mso-bookmark: _Toc243890372"><FONT size=5><FONT face=Calibri>Transformation of chemically competent <I style="mso-bidi-font-style: normal">E. coli</I></FONT></FONT></SPAN></SPAN></SPAN></SPAN></SPAN></A></SPAN><I style="mso-bidi-font-style: normal"><o:p></o:p></I></H2> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Defrost the frozen cells on ice. Use 50μl cells pr transformation for transformation of plasmids and 100 μl of cells for transformations of ligation mix. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Add an appropriate volume of plasmid</FONT><A title="" style="mso-footnote-id: ftn1" href="#_ftn1" name=_ftnref1><SPAN class=MsoFootnoteReference><SPAN style="mso-special-character: footnote"><SPAN class=MsoFootnoteReference><SPAN style="FONT-SIZE: 10pt; LINE-HEIGHT: 115%; FONT-FAMILY: 'Calibri','sans-serif'; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'Times New Roman'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-bidi-language: EN-US; mso-ansi-language: EN-US; mso-fareast-language: EN-US"><U><FONT color=#0000ff>[1]</FONT></U></SPAN></SPAN></SPAN></SPAN></A><FONT face=Calibri size=2> (often 1μl) to 50μl cell suspension. If transforming with a ligation mix add half of the ligation mix (5-10 μl ligation mix) to the 100μl cells.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Keep on ice for 30 minutes</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Heat chock for 90 seconds at 42°C.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Transfer quickly to ice and keep them for 5 minutes.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Plate 100 μl cell suspension on selective medium (LB-amp) and incubate at 37°C O/N. Store the rest of the transformed cells in the fridge. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Between 5 and 6 the following can be done:</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Add 1ml LB medium. Use 0,5 ml for ligation mixtures. Mix. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Incubate samples for 1 hour at 37°C.</FONT></P> <H2 style="MARGIN: 12pt 0cm 4pt"><A name=_Toc243890373></A><A name=_Toc235693650></A><A name=_Toc235602407></A><A name=_Toc220487574></A><A name=_Toc219878406></A><A name=_Ref219527576></A><A name=_Toc219522917></A><A name=_Toc188265038></A><A name=_Toc184047196><SPAN style="mso-bookmark: _Toc188265038"><SPAN style="mso-bookmark: _Toc219522917"><SPAN style="mso-bookmark: _Ref219527576"><SPAN style="mso-bookmark: _Toc219878406"><SPAN style="mso-bookmark: _Toc220487574"><SPAN style="mso-bookmark: _Toc235602407"><SPAN style="mso-bookmark: _Toc235693650"><SPAN style="mso-bookmark: _Toc243890373"><FONT face=Calibri size=5>High Efficiency Yeast Transformation</FONT></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></SPAN></A></H2> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><A name=_Toc219522918><STRONG><FONT face=Calibri size=2>DAY 1</FONT></STRONG></A><o:p></o:p></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Inoculate the yeast strain in 2-5ml of liquid YPD and incubate O/N at 30°C on a rotary shaker. Leave a 50 mL shakeflask in the incubater with YPD, so it will be 30 C in the morning.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>In the morning, inoculate a shake flask containing 50ml YPD with the 2-5ml cultures. Incubate 3-5 h at 30°C on a rotary shaker. (Option: take 1 mL of o/n culture and leave it for 5 hours)</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>NB: For a transformation with a basic plasmid, the O/N culture is plenty.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><A name=_Toc219522919><B style="mso-bidi-font-weight: normal"><FONT face=Calibri size=2>DAY 2</FONT></B></A><B style="mso-bidi-font-weight: normal"><o:p></o:p></B></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Harvest the cells by centrifugation at 3000g for 5 min (for the centrifuge in 220 this is equal to 4220 rpm). You need the rotor F-16 from the first floor that can take falcon tubes. The rotor code should be 30.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Wash the cells in 25ml sterile water. Centrifuge at 3000g for 5 min and resuspend cells in 1ml water.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells. <I style="mso-bidi-font-style: normal">It is not necessary to boil the carrier DNA every time. Keep the aliquot in freezer and boil again after 3-4 freeze-thaws. </I>(They can usually be found in lab 015)</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Transfer the cell suspension to a 1.5ml micro centrifuge tube and centrifuge for 30s. Remove the supernatant with a micropipette. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Resuspend the cells to a final volume of 1ml with water and vortex vigorously to resuspend the cells. Put the cells on ice. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Pipette 100µl samples (10<SUP>8</SUP> cells) into 1.5ml micro centrifuge tubes, one for each transformation and one extra for each culture for SC plating. Centrifuge at top speed for 30s and remove the supernatant with a micropipette. (NB: Remember to set water baths at 42).</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Make sufficient Transformation Mix corresponding to the number of transformations that need to be performed (count 1 extra). Keep the mix on ice/water.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="mso-bidi-language: AR-SA; mso-no-proof: yes"><v:shape id=Billede_x0020_2 style="VISIBILITY: visible; WIDTH: 486.75pt; HEIGHT: 157.5pt; mso-wrap-style: square" type="#_x0000_t75" o:spid="_x0000_i1026"><v:imagedata o:title="" src="file:///C:\DOCUME~1\Mads\LOCALS~1\Temp\msohtmlclip1\01\clip_image007.emz"><FONT face=Calibri size=2></FONT></v:imagedata></v:shape></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT size=2><FONT face=Calibri><SPAN style="mso-spacerun: yes"> </SPAN><I>All volumes are mentioned in µl<o:p></o:p></I></FONT></FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>(for a deletion you will need approx 300-500 ng of each fragment)</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><o:p><FONT face=Calibri size=2> </FONT></o:p></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l0 level1 lfo2"><SPAN style="FONT-FAMILY: Symbol; mso-fareast-font-family: Symbol; mso-bidi-font-family: Symbol"><SPAN style="mso-list: Ignore"><FONT face=Symbol size=2>·</FONT><SPAN style="FONT: 7pt 'Times New Roman'">        </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Place Xµl of plasmid on top of the cells. Add (360-X)µl of Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="FONT-SIZE: 5pt; LINE-HEIGHT: 115%"><o:p><FONT face=Calibri> </FONT></o:p></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l0 level1 lfo2"><SPAN style="FONT-FAMILY: Symbol; mso-fareast-font-family: Symbol; mso-bidi-font-family: Symbol"><SPAN style="mso-list: Ignore"><FONT face=Symbol size=2>·</FONT><SPAN style="FONT: 7pt 'Times New Roman'">        </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Incubate the tubes at 42°C for 40 min</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="FONT-SIZE: 5pt; LINE-HEIGHT: 115%"><o:p><FONT face=Calibri> </FONT></o:p></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l0 level1 lfo2"><SPAN style="FONT-FAMILY: Symbol; mso-fareast-font-family: Symbol; mso-bidi-font-family: Symbol"><SPAN style="mso-list: Ignore"><FONT face=Symbol size=2>·</FONT><SPAN style="FONT: 7pt 'Times New Roman'">        </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Micro centrifuge at top speed for 30s and remove the transformation mix with a pipette.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="FONT-SIZE: 5pt; LINE-HEIGHT: 115%"><o:p><FONT face=Calibri> </FONT></o:p></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l0 level1 lfo2"><SPAN style="FONT-FAMILY: Symbol; mso-fareast-font-family: Symbol; mso-bidi-font-family: Symbol"><SPAN style="mso-list: Ignore"><FONT face=Symbol size=2>·</FONT><SPAN style="FONT: 7pt 'Times New Roman'">        </SPAN></SPAN></SPAN><FONT face=Calibri size=2>Redissolve in 200µl of sterile water into each tube. Stir the pellet by pipetting and vortex.</FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="FONT-SIZE: 5pt; LINE-HEIGHT: 115%"><o:p><FONT face=Calibri> </FONT></o:p></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt 18pt; TEXT-INDENT: -18pt; tab-stops: list 18.0pt; mso-list: l0 level1 lfo2"><SPAN style="FONT-FAMILY: Symbol; mso-fareast-font-family: Symbol; mso-bidi-font-family: Symbol"><SPAN style="mso-list: Ignore"><FONT face=Symbol size=2>·</FONT><SPAN style="FONT: 7pt 'Times New Roman'">        </SPAN></SPAN></SPAN><FONT face=Calibri size=2>For transformation with an integrative plasmid, linear construct or nucleotide, plate 100µl onto 2 selective plates. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><SPAN style="FONT-SIZE: 5pt; LINE-HEIGHT: 115%"><o:p><FONT face=Calibri> </FONT></o:p></SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><FONT face=Calibri size=2>Incubate the plates at 30°C for 2-3 days. </FONT></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 10pt"><o:p><FONT face=Calibri size=2> </FONT></o:p></P> <P class=MsoNormal style="MARGIN: 24pt 0cm 10pt"><o:p><FONT face=Calibri size=2> </FONT></o:p></P> <DIV style="mso-element: footnote-list"><BR clear=all><FONT face=Calibri size=2> <HR align=left width="33%" SIZE=1> </FONT> <DIV id=ftn1 style="mso-element: footnote"> <P class=MsoFootnoteText style="MARGIN: 0cm 0cm 10pt"><A title="" style="mso-footnote-id: ftn1" href="#_ftnref1" name=_ftn1><SPAN class=MsoFootnoteReference><SPAN style="mso-special-character: footnote"><SPAN class=MsoFootnoteReference><SPAN style="FONT-SIZE: 10pt; LINE-HEIGHT: 115%; FONT-FAMILY: 'Calibri','sans-serif'; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'Times New Roman'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-bidi-language: EN-US; mso-ansi-language: EN-US; mso-fareast-language: EN-US"><U><FONT color=#0000ff>[1]</FONT></U></SPAN></SPAN></SPAN></SPAN></A><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><FONT size=2><FONT face=Calibri> Approximately 50ng should be added if transforming with a plasmid. <o:p></o:p></FONT></FONT></SPAN></P></DIV></DIV>

<td width="182px" height="100%" valign="top" class="greybox"> <font face="arial" size="2"> Work process

Our team works parallel in smaller sub-teams. Some of us work hard in the lab, while others are in the process of developing software and the in silico model of the Redoxilator system. However, we constantly keep each other updated, and meet often to exchange ideas and take turns at the different tasks, thus exhausting all of our combined knowledge in every aspect of this project.