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RecA: Week 8, July 3-9
From 2011.igem.org
Contents |
Sunday, July 3
Insert RecAI into K3 Vector, Take 3 Day 3
nbsp;nbsp;nbsp;nbsp;The K3 plasmid was extracted using the Omega Bio-Tek miniprep kit. The plasmid was digested with XbaI and SpeI. No band showed on an agarose gel, indicating the lack of DNA. The assembly could not be performed until the plasmid extraction problems get fixed. The RecAI PCR products were prepared from genomic DNA.
Tuesday, July 5
RecAI Mutagenesis Test Take 4, Day 1
The terminator (B0015) and the driver (I763007) plasmids from the registry kit were transformed into Escherichia coli cells.
Insert RecAI into K3 Vector, Take 4 Day 1
The RecAI PCR product was purified and digested. The K3 plasmid was also digested. These parts were ligated together and transformed into Escherichia coli cells. The recovery media was plated onto kanamycin resistant plates.
Wednesday, July 6
RecAI Mutagenesis Test, Take 4 Day 2
The terminator (B0015) and the driver (I763007) cells did not grow in the cultures. The plasmids from the registry were tranformed again into Escherichia coli cells and put into culture to grow overnight.
Insert RecAI into K3 Vector, Take 4 Day 2
The cloning attempt from 7/5 was verified through gel electrophoresis. The RecAI looked like the correct size, so the colonies were grown up in culture for sequencing tomorrow.
Thursday, July 7
nbsp;nbsp;nbsp;nbsp;The cultures from 7/6 were accidentally placed into the 50°C incubator instead of the 37°C. The cultures were placed in the correct incubator once the mistake was discovered, but the cells may have died. The plasmids were extracted anyway.
RecAI Mutagenesis Test, Take 4 Day 3
The registry plasmids were transformed a third time into Escherichia coli cells.
Insert RecAI into K3 Vector, Take 4 Day 3
The sequencing results came back and appeared to be correct. The sequencing read was not very clear, so the plasmid was extracted again and sent to sequencing in an attempt to get a better read for clearer results. The mutagenesis process will start tomorrow, even before the new sequencing results arrived.
Friday, July 8
RecAI Mutagenesis, Day 1
The mutagenesis protocol found on the Richard Lab openwetware page (developed by Jim and Brian) was followed. The first mutagenesis was performed to eliminate the EcoRI and PstI sites from the RecAI gene. The extension time for the PCR amplification was 2:15. The PCR products digested with DpnI for five hours. The PCR purification process was performed incorrectly, contaminating the PCR products. The purification process was continued anyway in case the contamination was not harmful.
RecAI Mutagenesis Test, Take 5 Day 1
The terminator (B0015) and driver (I763007) plasmids were extracted using the Omega Bio-Tek miniprep kit. The driver insert was amplified through PCR and the PCR products were purified.
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