(→CeaB and CeiB transformation into plasmid pSBA3)
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''Marcelo''
''Marcelo''
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==== PY Promoter - Digestion and ligation reactions ====
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*<p style=”text-align:justify;”>Today we digested PY1 + pGEM with EcoRI and SpeI to excise PY fragment.</p>
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*<p style=”text-align:justify;”>We also digested the plasmid BBa_J23100 (that one with the RFP reporter) with EcoRI and SpeI. With this plasmid digested with this 2 enzymes it is going to be possible to insert our digested PY fragment into this plasmid.</p>
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==== CeaB and CeiB transformation into plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3] ====
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*<p style=”text-align:justify;”>This digestion reactions lasted for 3 hours.</p>
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*<p style=”text-align:justify;”>Today, we dephosphorilated the plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3], and immediately started the ligation reaction of this unphosphorylated plasmid with CeaB and CeiB. Considering that the dephosphorylation use buffer that contains salt and that salt could be interfering in the transformations, we performed a dyalisis to get the samples free of salts. At last, we transformed competent ''E. coli'' (according to the [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation protocol 3]) and plated the respective plates. This time we expect to obtain transforming cells.</p>
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*<p style=”text-align:justify;”>After the digestion we performed the ligation reaction of PY1 + BBa_J23100 following [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11].</p>
Purification of pTet and double terminator biobricks
Once we digested those biobricks yesterday, we could proceed today to the purification of the target band from an agarose gel.
We used Invitrogen's Purelink Quick Gel Extraction Kit, following the manufacturer's protocol (Protocol 7), without modifications.
Marcelo
CeaB and CeiB transformation into plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3]
Today, we dephosphorilated the plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3], and immediately started the ligation reaction of this unphosphorylated plasmid with CeaB and CeiB. Considering that the dephosphorylation use buffer that contains salt and that salt could be interfering in the transformations, we performed a dyalisis to get the samples free of salts. At last, we transformed competent E. coli (according to the protocol 3) and plated the respective plates. This time we expect to obtain transforming cells.
Luige
YeastGuard
Dephosphorylation - CIAP test
The E.coli didn´t grow in any of the plates again. =[ We discovered that the electrocompetent E.coli that we used yesterday were dead! So we transformed another electrocompetent E.coli using the rest of the ligation reactions we had. We extremely hope to find colonies tomorrow!
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