"
RecA: Week 6, June 19-24
From 2011.igem.org
Contents |
Sunday
RecAI Mutagenesis Test Take 2, Day 12
Once again, the plates held no colonies, meaning that the assembly did not work and must be performed again.
Monday
RecAI Mutagenesis Test Take 2, Day 13
Since the entire lab is having trouble with assembly, the validity of the restriction enzymes, especially PstI, has been called into question. The reporter group ran digests using XbaI and AgeI as well as XbaI and PstI, and these digests were run on a 1% agarose gel. The old RecA digests from 6/17 were also ran on the gel. The terminator (B0015) and the PcI reporter (I763007) were miniprepped.
RecAI Extraction Take 6, Day 1
The purified RecAI PCR product from 6/14 was digested with the EcoRI and SpeI enzymes in buffer two. The K3 miniprep from 6/17 was digested with EcoRI and SpeI in buffer two. These restriction enzymes, however, were not appropriate for this assembly, so the assembly was discontinued.
Tuesday
The sensor group test all of the restriction enzymes by digesting K3 with EcoRI, EcoRI + XbaI, SpeI + PstI, EcoRI + PstI, XbaI + SpeI. These digests were run on a 1% agarose gel in order to see if the restriction enzymes were still alive and cutting the plasmid correctly. The gel showed that the enzymes still worked, so the failed assemblies have a different cause.
RecAI Mutagenesis Test Take 2, Day 14
The PcI reporter (I763007) was amplified through PCR and then purified.
RecAI Extraction Take 6, Day 2
The K3 miniprep from 6/17 and the purified RecAI PCR product from 6/14 were digested with XbaI and PstI. Gel elecrophoresis on the K3 vector was performed, and the band around 4000 base pairs was extracted. This gel extraction was purified using the kit from Omega Bio-Tek. The resulting product (K3) was ligated with RecAI, and the ligation was transformed into Escherichia coli cells and plated onto kanamycin resistant plates.
Wednesday
RecAI Mutagenesis Test Take 2, Day 15
The terminator (B0015) and PcI reporter (I763007) were assembled together again through amplified insert assembly.
RecAI Extraction Take 6, Day 3
The RecAI did not grow on the plate again. When the sequencing of RecAI was analyzed, a PstI site was found. This could cause the primers to bind to the wrong location because the PstI restriction enzyme would cut in all PstI active sites, one of which is in the middle of the RecAI gene. Therefore, future assemblies will use XbaI and SpeI and XbaI and PstI. Even though the wrong restriction enzymes were used for assembly, colonies still should have grown on the plates. Thus, a member of another group will supervise the next insertion of RecAI into the K3 vector.
Thursday
RecAI Mutagenesis Test Take 2, Day 16
The assembly performed 6/22 resulted in colonies, so colony PCR amplified the colonies. The PCR product was run on a 1% agarose gel and the results showed that the assembly contained the correct number of base pairs. Sequencing will further verify the assembly's validity.
RecAI Extraction Take 7, Day 1
The
