Team:Warsaw/Calendar-Main/28 August 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Amplification of the mgtc promoter using new primers pSB1A3 plasmid digest 

Methods:  The PCR mix was prepared as follows: 5&mu;l buffer B (EURx) 2&mu;l 5mM dNTPs (EURx) 5&mu;l forward starter 5&mu;l reverse starter 2&mu;l OptiTaq polymerase (EURx) 2&mu;l Salmonella matrix 29 &mu;l H2O  PCR programme: 4min 95&deg;C (30s 95&deg;C, 35s 48&deg;C, 40s 72&deg;C)x28 (30s 95&deg;C, 35s 58&deg;C, 40s 72&deg;C)x28 10min 72&deg;C ~ 4&deg;C  The PCR results were visualised with gel electrophoresis on 1% agarose gel. The digest mix was prepared as follows: 60&mu;l plasmid isolation 7&mu;l Buffer O (Fermentas) 1&mu;l EcoRI enzyme 1&mu;l PstI enzyme 1&mu;l H2O  The digest was carried out in 37&deg;C for 3h and inactivated for 15 min. in 80&deg;C. 1&mu;l of CIAP enzyme was added after the inactivation and allowed to work for 1h in 37&deg;C and was later inactivated for 15 min. in 85&deg;C.</li> The resulting mix was separated on 1% agarose gel alongside the PCR and then selected bands were extracted for purification.</li> </ul>

Results:  No gel picture, but the PCR worked so great that I decided not to irradiate the gel with UV light more than it was necessary for band extraction. </li> </ul>

Conclusions:  The temperatures were chosen... wisely.</li> </ul>

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Cro-box digest</li> </ul>

Methods:  The digest mix was prepared as follows: 20&mu;l plasmid isolation 3&mu;l Buffer O (Fermentas) 1&mu;l EcoRI enzyme 1&mu;l PstI enzyme 5&mu;l H2O </li></ul>

Assembly of endosomal detection operon Marcin Task 1:  Gel-outs of following constructs digested on <a href:"http://2009.igem.org/Team:Warsaw/Calendar-Main/27_August_2009">17.08.09</a>:</li> <a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</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> <a href="http://partsregistry.org/Part:BBa_E0022"> BBa_E0022</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></ul> Methods:  <li>All gel-outs were performed using the EurX gel-out kit according to the manual</a></li></ul> Results: <ul><li>Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation</ul></li>  Comment: Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid was insufficient to perform gel-out. It is obligatory to digest this construct one more time Task 2: <ul><li>Digestion of following construct:</li></ul> <ul><li><a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</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> Methods: <ul><li>Reaction mixture composition:</li> 20 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) 1 &mu;l PstI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 15 &mu;l MQ water <li>Digest was performed about six hours and subsequently thermal inactivated</li></ul> Task 3: <ul> <li>Gel-outs of construct described in Task 2</li> </ul> Methods: <ul> <li>All gel-outs were performed using the EurX gel-out kit according to the manual</a></li></ul> Results: <img src="http://2009.igem.org/wiki/images/f/ff/E0022%2BRBS_C0051%2BRBS_digest_28_08_09.png" width="20%" heigth="20%"> <font face="Times New Roman" size="3"> The picture reveal low yield of the gel-out <ul><li>Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation</ul></li>  Comment: Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid may be insufficient for efficient gel-out. Although I decided to continue the task Task 4: <ul><li>Cloning <a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:BBa_E0022"> BBa_E0022</a> ligated with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> vector</ul></li> Methods: <ul> <li> Ligation mixtures composition:</li> 30 &mu;l digested insert 10 &mu;l digested vector 5 &mu;l Tango buffer(Fermentas) 3 &mu;l dNTPs mixture (EurX, concentration 5 mM) 2 &mu;l ligase T4 (Fermentas) <li>Duration of ligation was about 18 hours; reaction was conducted in 19 &deg;c (approximately).</li> <li>In the next step ligated samples were thermally inactivated via heating in 80 &deg;c for 20 minutes</li> </ul>

Making of the plac-RBS-llo-intA part Jarek

Tasks: <ul> <li>Digestion of pUC19-intA with PstI and CaiI endonucleases, and plac-RBS-llo with PstI (this sample was additionaly defosforylated during digestion).</li> <li>Separation of digested fragments in 0,8% agarose gel</li> <li>Another digestion of pUC19-intA and plac-RBS-llo with the same enzymes</li> </ul> Results: <ul> <li>After seting electrophoresis I found out that the digestion was prepared wrong, also gel went wrong. </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>