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RecA: Week 7, June 26-July 2
From 2011.igem.org
Contents |
Sunday, June 26
Insert RecAI into K3 Vector, Day 1
The RecAI PCR product was purified, then digested with SpeI and XbaI. The K3 vector plasmid (extracted on 6/24) was digested with SpeI and XbaI as well. The digests were ligated together, the ligation was transformed into Escherichia coli cells, and the cells were plated onto kanamycin resistant plates.
Monday, June 27
RecAI Mutagenesis Test Take 2, Day 19
The sequencing results from Friday, 6/24, showed the terminator (B0015) twice without the cI reporter (I763007). The miniprep from 6/20 and from the registry were amplified through PCR. The terminator (B0015) from Mike Speer's stock was grown in culture. This stock was used so that the RecA group would know that it was not contaminated or mislabeled.
RecAI Extraction Take 7 Day 4
The sequencing results for the RecAI + K3 plasmid showed that the vector did not take the insert, but instead closed in on itself. The RecAI colony from 6/26 was amplified through colony PCR. The gel electrophoresis did not yield any bands. The K3 and RecAI digests were ligated together again.
Tuesday, June 28
RecAI Mutagenesis Test Take 3, Day 1
The terminator (B0015) was extracted from culture through the Omega Bio-Tek miniprep protocol. The miniprep was digested with PstI and SpeI. The registry miniprep as well as the PCR product of the 6/20 miniprep were run on a gel, which showed that both were correct. Thus, the miniprep is labeled correctly. The registry PCR product was purified and digested with XbaI and PstI. The digests were ligated together, transformed into Escherichia coli cells, then plated onto an antibiotic resistant plate.
Insert RecAI into K3 Vector, Day 2
Five colonies containing the RecAI + K3 plasmid were amplified through colony PCR.
Wednesday, June 29
RecAI Mutagenesis Test Take 3, Day 2
Colonies from the assembly performed on 6/28 were amplified through colony PCR. The results were run on an agarose gel. The gel contained bands around 1000 base pairs, indicating a successful assembly. The colonies were grown up in culture overnight.
Thursday, June 30
RecAI Mutagenesis Test Take 3, Day 3
The plasmids of the cells grown up on 6/29 were extracted using the Omega Bio-Tek miniprep kit. These plasmids were sent to the sequencing center for verification. The sequencing results came back inconclusive. As a result, the assembly will have to be repeated.
Friday, July 1
Insert RecAI into K3 Vector, Take 2 Day 1
The PCR purified RecAI from 6/20 was digested with XbaI. The digest was heat killed and left in the 4°C overnight because we did not have any K3 plasmid left.
Saturday, July 2
Insert RecAI into K3 Vector, Take 3 Day 2
The K3 plasmid was extracted from the cells that grew overnight. The miniprep was digested with XbaI and SpeI. The digests (including the one from 7/1) were run on an agarose gel. The gel showed no bands, indicating a poor miniprep. More cells containing K3 were grown up overnight.
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