Team:Warsaw/Calendar-Main/25 September 2009

PCR PhoP and PhoQ Kama

Tasks:  Amplification of phoP and phoQ 

Methods:  PCR mixture's composition: 1&mu;l Pfu buffer (EURX) 0,25&mu;l primer phoPF for phoP and phoQF for pho Q 0,25&mu;l primer phoPR for phoP and phoQR for pho Q 0,5&mu;l dNTPs (10 mM) 0,25&mu;l Pfu turbo polymerase (EURX) 0,5&mu;l template DNA from Salmonella (Salmonella enterica s. Thypimurium LT2) 0,75&mu;l DMSO The solution was topped up with H2O to 10&mu;l.    PCR programs: phoP 4min 95&deg;C (30s 95&deg;C, 1min 60&deg;C, 1min 40s 72&deg;C)x35 10min 72&deg;C ~ 7&deg;C   phoQ 4min 95&deg;C (30s 95&deg;C, 1min 60&deg;C, 3min 10s 72&deg;C)x35 10min 72&deg;C ~ 7&deg;C   Electrophoretic separation on 1% agarose gel</li> </ul>

Results: <img src="http://2009.igem.org/wiki/images/c/c7/2009_09_25_phoP_phoQ_opisane.JPG"/>  Gel (from left)</li> </ul> <ol> 1-3 controls -</li> 4 - phoP</li> 5 - phoQ</li> 6-7 - not important (phoP/phoQ for gel-out)</li> M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> </ol> Notes:  </b></li> </ul>

Assembly of fusion proteins Marcin

Task 1: Amplification of p53 (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition: 5 &mu;l Pfu buffer (EURX) 2 &mu;l Pfu turbo polymerase (EURX) 5 &mu;l dNTPs (EURx, 5 mM) 0,1 &mu;l template DNA (isolated plasmid) 1 &mu;l primer p53silverF 1 &mu;l primer p53silverR 36 &mu;l MQ water

PCR program: 1. 98&deg;C - hold 2. 98&deg;C - 2 minutes 3. 98&deg;C - 15 sec 4. 64&deg;C - 30 sec 5. 68&deg;C - 2 minutes go to 2 until number of cycles=35 6. 68&deg;C - 10 minutes 7. 4&deg;C - hold

Task 2: Amplification of bax (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition: 5 &mu;l Pfu buffer (EURX) 2 &mu;l Pfu turbo polymerase (EURX) 5 &mu;l dNTPs (EURx, 5 mM) 0,1 &mu;l template DNA (isolated plasmid) 1 &mu;l primer bax_silverF 1 &mu;l primer bax_silverR 36 &mu;l MQ water

PCR program: 1. 98&deg;C - hold 2. 98&deg;C - 2 minutes 3. 98&deg;C - 15 sec 4. 70&deg;C - 30 sec 5. 68&deg;C - 1.5 minutes go to 2 until number of cycles=30 6. 68&deg;C - 5 minutes 7. 4&deg;C - hold

Task 3: Amplification of signal peptide from COX (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition: 5 &mu;l Pfu buffer (EURX) 2 &mu;l Pfu turbo polymerase (EURX) 5 &mu;l dNTPs (EURx, 5 mM) 0,1 &mu;l template DNA (isolated plasmid) 1 &mu;l primer mitoCOXsilverF 1 &mu;l primer mitoCOXsilverR 36 &mu;l MQ water

PCR program: 1. 98&deg;C - hold 2. 98&deg;C - 2 minutes 3. 98&deg;C - 15 sec 4. 69.3&deg;C - 30 sec 5. 68&deg;C - 45 sec go to 2 until number of cycles=30 6. 68&deg;C - 5 minutes 7. 4&deg;C - hold

Task 4: Amplification of YFP (ready to fusion in Silver Standard) via PCR reaction

Methods: Reaction mixture composition: 5 &mu;l Pfu buffer (EURX) 2 &mu;l Pfu turbo polymerase (EURX) 5 &mu;l dNTPs (EURx, 5 mM) 0,1 &mu;l template DNA (isolated plasmid) 1 &mu;l primer YFPsilverF 1 &mu;l primer YFPsilverR 36 &mu;l MQ water

PCR program: 1. 98&deg;C - hold 2. 98&deg;C - 2 minutes 3. 98&deg;C - 15 sec 4. 70&deg;C - 30 sec 5. 68&deg;C - 1.5 minutes go to 2 until number of cycles=30 6. 68&deg;C - 5 minutes 7. 4&deg;C - hold

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Plasmid isolation</li> Control digest</li> </ul>

Methods:  The plasmids were isolated from 3&mu;l of overnight liquid cultures using the Plasmid Mini kit (A&A Biotechnology). The digest mix was prepared as follows: 20&mu;l purified plasmid 3&mu;l Orange Buffer (Fermentas) 1&mu;l PstI Enzyme (Fermentas) 1&mu;l EcoRI Enzyme (Fermentas) 5&mu;l H2O </li> The digest was carried out in 37&deg;C for 4h.</li> <li>The results were visualized on 2% agarose gel.</li> </ul>

Results: <ul> <img src="http://2009.igem.org/wiki/images/3/3d/2009.09.25_-_cro-box.jpg"> <li>Gel (from left):</li> <ol> <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> <li>Sample A</li> <li>Sample B</li> <li>Sample C</li> <li>Sample D</li> <li>Sample E</li> <li>Reference sample</li> <li>Sample F</li> <li>Sample G</li> <li>Sample H</li> <li>Sample I</li> </ol> </ul> Conclusions: <ul> <li>Sample F looks like a hit. It was sent to be sequenced.</li> </ul>

[UPDATE:] The nucleotides in sample F read: "TATCCCTTGCGGTGATA" (prefix and suffix omitted).