Laboratory Notebook

06-08-2012 to 12-08-2012

Mutagenesis on sequenced FFT and SST

Today our primes arrived. We did a mutagenesis on SST and FFT to correct some genetic errors which our sequencing results showed us. Next we tranformed bacteria with SST and FFT.
The SST plates that had been grown over night showed some colonies that were selected and put into liquid LB. for overnight culture. The FFT plates showed no colonies.
we tried again to run a mutagenesis on the FFT genes, this time following the protocol that was delivered with the mutagenesis kit. next we transformed XL10-GOLD e.coli with the mutated genes and plated them out on agar plates with ampicilin, and left ON. in the incubator.

FFT plates from the day before showed colonies and were transferred to liquid medium and incubated overnight. Plasmid SST DNA from liquid cultures from the day before were purified, digested with EcoRI, SpeI, XbaI and PstI, then run on a gel(picture not included). Results were awesome.
EcoRI+PstI: 1-8
Ladders: 9-10
XbaI+SpeI: 11-18
Colony 1 and 2 were most awesome. Will be shipped for sequencing later on.

The plasmid DNA from the liquid cultures of FFT from the day before were purified, digested with EcoRI, SpeI, XbaI and PstI, then run on a gel. While the gel was running, appropriate values of expected lengths were calculated:
EcoRI+SpeI: 2000bp+3000bp WRONG:200bp+1800bp+3000bp or 1length
XbaI+PstI: 2000bp+400+2600bp WRONG: 1200bp+800bp+400bp+2600bp or 1length or 800bp+400bp+3800bp
On the gel, the XbaI+PstI is seen on the left, EcoRI+SpeI is seen on the right
The results from the gel:
The conclusion from the gel is that the illegal XbaI site is removed, the desired XbaI site is present, and.... unfortunately almost all the plasmids still contain the illegal EcoRI site... except either of colony 3 and 4, which were mixed by accident the day before, and can be found at position 3 from the left on the gel. Beforehand, we expected trouble with the Primer designed to remove the EcoRI site, because it had a lot of problematic alignments(see sequence).
Following, another two batches of liquid colony was prepared (from the remains of colony 3 and 4) and incubated overnight, also another two plates were prepared as a safety backup with the mixed liquid medium of the two colonies.

The backup plates showed immense growth. They will be kept at 4C and saved for later use, if the liquid cultures fail. The liquid cultures of FFT colonies 3 and 4 from the day before were purified, digested with EcoRI+SpeI and XbaI+PstI, then run on a gel.
The results:
In conclusion: though faint, colony 4 shows signs of the illegal EcoRI restriction site. It is easier to see on a picture taken on the gel from earlier, which can be found in the Lab. No digital copy was made from the early gel photo.
Meanwhile, colony 3 is awesome. It will be prepared and sent for sequencing monday.