Team:Warsaw/Calendar-Main/20 August 2009

Amplyfing of Pho sequence Justyna Methods  PCR reaction mix: per 50μl: 5.0 μl - 10 x buffer (20mM MgSO4) 3.5 μl - dNTP mix (5mM) 2.5 μl - primer PhoF 2.5 μl - primer PhoR2 3.75μl - DMSO 2.0 μl - template DNA 28.25μl - mQ water 2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl) buffer, dNTP mix and polymerase from EURx. DNA template - Salmonella genomic DNA  PCR reaction has been done in two extension temperatures - 68°C and 72°C  Thermal cycling conditions for PCR: 94°C, 1 min 0s

94°C, 0 min 15s 55°C, 0 min 30s 68°C / 72°C (either), 1 min 0s cycles 1-25

68°C / 72°C (either), 7 min 0s 4°C, indefinite Results: Effective reaction.   Cloning Pho into pSB1A3 plasmid Justyna Task 1:  Gel-out Pho PCR product</ul> Methods: Pho PCR products were isolated from agarose gel using A&A Gel-Out kit.</li> <img src="http://2009.igem.org/wiki/images/3/39/Phoafergeloutopis.jpg"> The arrow points the right and best isolated product</li> </ul> pSB1A3 plasmid was previously prepared as described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/14_August_2009">here</a> </li>

</ul> </ul>

Assembly of endosomal detection operon Marcin Task 1: Gel-out of <a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</a> with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li></ul> Methods:  sample was inactivated via heating in 80 &deg;C for 20 minutes Gel-out was performed using the EurX gel-out kit according to the manual</a></li></ul> Results: <img src="http://2009.igem.org/wiki/images/5/5c/C0040%2BRBS_20_08_09.png" width="25%" heigth="25%" <font face="Times New Roman" size="3"> Result of the digestion Task 2:  Ligate <a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</a> with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> to <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid with <a href="http://partsregistry.org/Part:BBa_R0080"> BBa_R0080</a> </li></ul> Methods:  Reaction mixture composition:</li> 10 &mu;l insert 7 &mu;l vector 2.3 &mu;l Buffer Tango (Fermentas) 3 &mu;l dNTPs mixture (EurX) 1 &mu;l T4 ligase (Fermentas) Ligation was performed for about 12 hours and it was subsequently thermally inactivated</li></ul> Task 3: <li>Transformation of chemocompetent E. coli strain DH5&alpha</ul></li> Constructs to transform: </ul> <ul> <li>p53 CDS + <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid </li> <li>cro CDS + <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li></ul> Methods: <ul> <li>Detailed protocol of transformation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>.</li> </ul>

Making of the plac-RBS-llo part Jarek

Tasks: <ul> <li>Separation of restriction fragments in 0,8% agarose gel </li> <li>Digestion of 5 samples of plasmid DNA acquired on 19 August with PvuII and PstI restriction endonucleases</li> </ul>

Cloning switch 1 regulatory parts [ <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>-PcI.RBS.LacI, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>-PcI.RBS.LacI.PcI.RBS.RFP.terminator, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>-PLacI.RBS.cI.terminator, <a href="http://partsregistry.org/Part:BBa_K177038">K177038</a>-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator ] into two compatible low copy number plasmids of different antibiotic resistance Ania Tasks: <ul> <li> </li> </ul>