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Team:Penn State/Notebook
From 2014.igem.org
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<td><center><b>Friday, June 6, 2014</b></center></td> | <td><center><b>Friday, June 6, 2014</b></center></td> | ||
<td>Conducted colony PCR using <i>P. putida</i> KT2440 strain as the DNA template to construct 1 kb overlaps for homologous recombination. All four of the first genome overlaps with gene PP_0747 were successful; only 2 overlaps with <i>upp</i> gene were successful. These were gel purified. We learned Gibson Chew-Back Annealing Assembly (CBA) protocol. </td> | <td>Conducted colony PCR using <i>P. putida</i> KT2440 strain as the DNA template to construct 1 kb overlaps for homologous recombination. All four of the first genome overlaps with gene PP_0747 were successful; only 2 overlaps with <i>upp</i> gene were successful. These were gel purified. We learned Gibson Chew-Back Annealing Assembly (CBA) protocol. </td> | ||
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<td><center><b>Sunday, June 8, 2014</b></center></td> | <td><center><b>Sunday, June 8, 2014</b></center></td> | ||
| - | <td></td> | + | <td>One 4-part, two 3-part, and two 2-part Gibson CBA's were conducted to assemble the kanamycin resistance cassette, two genome overlaps, and the colE1 replication origin. </td> |
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<td><center><b>Monday, June 9, 2014</b></center></td> | <td><center><b>Monday, June 9, 2014</b></center></td> | ||
| - | <td></td> | + | <td>The two 2-part and two 3-part CBA's were amplified using PCR Rescue and gel purified. The original 4-part CBA was transformed into <i>E. coli</i> electrocompetent cells using electroporation and plated on kanamycin antibiotic agar plates. The 4-part CBA was repeated to ensure accuracy. Because the CBA parts contained no plasmid DNA, the 4-part CBA could be digested by restriction enzyme Dpn1. Dpn1 binds and cuts methylated DNA sites, thus destroying any plasmid DNA remaining as a contaminant.</td> |
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<td><center><b>Tuesday, June 10, 2014</b></center></td> | <td><center><b>Tuesday, June 10, 2014</b></center></td> | ||
| - | <td></td> | + | <td>The original 4-part CBA worked! Many colonies appeared on the plate after incubation at 37 degrees C for 18 hours, and 12 colonies were selected for plasmid preparation. These were digested with AatII and XbaI, two restriction sites that are only both contained in the final assembled 4-part plasmid. 6/12 colonies showed the correct bands on the gel. We also prepared more 1 kb ladder from concentrate.</td> |
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Revision as of 18:40, 20 June 2014
WELCOME TO iGEM 2014!Your team has been approved and you are ready to start the iGEM season!
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Penn State iGEM 2014 Notebook PageHere you will find weekly summaries of our wet laboratory progress, team updates, and accomplishments outside the laboratory. Below [link this] is our detailed, day-to-day progress laboratory notebook. Weekly Summarieswill create links to each week, but will just list down the page Week 1, Week 2, etc etc Laboratory Notebook
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