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Team:EPF-Lausanne/Notebook/Wet Lab
From 2009.igem.org
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Wet Lab
17.07.09
The plates and tubes from yesterday transformation weren't very concluant : almost no colony on the plates (actually we saw 3 of them but this might be contamination) and for the tubes there were almost no bacteria. We decided to do the miniprep with it anyways, to see what it gives :
Miniprep of LovTAP-Term, CT negative of the ligation and Term (that grew very well).
PCR of the miniprep products.
Gel electrophoresis of the PCR products and also of the plasmid of LovTAP-Term, CT neg and Term. We can see on the gel that there was actually no DNA on the LovTAP-Term and CT negative tubes.
In parallel, we began all over again, by taking Term from the kit. We used a different method, as the other one seems not to be working (the Term plasmid seems not to accept LovTAP in it). So this time we used the iGEM protocol to do the digestion. As the P site is in LovTAP, which is the upstream part, it is not a problem.
We did the digestion, ligation and transformation of LovTAP-Term and this time we really hope this will work.
18.07.09
Result of the transformation: nothing grew.
20.07.09
As the transformation of Saturday didn't work, we think that the plasmid Term BBa_B0010 has a problem. We tried to begin all over with 4 other Terminator from the registery.
We first did an extraction of 4 new Term plasmid from kit plate. Then we did a transformation of these 4 Term + a transf. with death cassette (with the special protocole for One Shot ccdB survival cells).
At the same time, we did a PCR with the 4 plasmids from the kit + LovTAP. We purified these PCR products, in the idea to do a digestion and then a ligation with them.
In parallel, we ran a gel with the Term plasmids, but there were no bands on the gel, so we didn't do the digestion. We will wait to have the transformation products to do that.
We also prepared LB-plates : Amp-Kana, Amp-Kana-Chl and Kana-Chl.
21.07.09
The transformation of the Term plasmids worked, so we did glycerol stocks of the 4 new Term and the death cassette.
Then miniprep of the 5 transformation products.
Digestion following the iGEM protocol : we cut with X and P Term, E and P the backbone and we take a LovTAP that was digested Friday with E and S.
Running of a gel to check the digestion products. Actually it seems the digestion didn't work. We did a second gel, by loading more product, and we clearly got the same result.
As the ligation this way seems not to work (either the enzymes for the digestion aren't working, either there is another problem in the ligation), we decided to try another way to do it : by doing a 2 step PCR. We designed and ordered the primers, now we just have to wait for them to go on in our cloning of LovTAP. Have a look at the cloning strategy to see how we designed the primers.
The idea of 2 step PCR : Media:2step pcr.pdf
22.07.09
We sent the miniprep product of LacI-RBS to sequencing. With all the problems we have with LovTAP-Term, we are really not sure that LacI-RBS really ligated well, so we want to have the sequence to check.
23.07.09
Plates with different bacteria concentrations were made to study which is the best concentration (amount) of bacteria we might let grow on plates.
Results of the sequencing of LacI-RBS : none... actually they weren't able to sequence it, it seems not to be pure enough. So we will do the ligation again today, including many control steps this time !
24.07.09
We found a Term plasmid that last year team used, and that worked : BBa-B0015.
We did 3 ligations in parallel :
LovTAP-Term with iGEM protocol and using the plasmid BBa-B0015.
LovTAP-Term using the Term digested last year with EX. (The idea is to insert LovTAP in the Term directly)
LacI-RBS with iGEM protocol again.
So we first did the digestion :
1. LovTAP cut with E and S, Term plasmid cut with X and P, the backbone cut with E and P.
2. LovTAP cut with E and S. We will use the Term from last year, already digested.
3. LacI with E and S, RBS with X and P and the backbone with E and P.
Next we ran a gel to check the digestion products.
We did the 3 ligations in parallel.
At this time we had the results of the gel, and actually the digestion didn't work. The backbones were digested but not the rest. There were no bands at all for the LovTAP and the others weren't digested. We will begin all over but using another protocol than iGEM's for the digestion
27.07.09
LOVTAP gene have been amplified again and runned on a gel to be sure to have the correct insert.
[http://partsregistry.org/Part:BBa_B0015 BBa_B0015 (terminator)] was digested, runned on a gel (which displayed the correct band) and extracted with the gel extraction kit, this step didn't worked well.
LOVTAP gene have been than digested, purified and runned on a gel.
We already had a digested [http://partsregistry.org/Part:BBa_B0015 BBa_B0015 (terminator)].
Finally we ligated overnight at 4°C the digested LOVTAP in both [http://partsregistry.org/Part:BBa_B0015 terminators], the one digested-extracted today and the one already digested-extracted.
