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Team:EPF-Lausanne/Notebook/Wet Lab
From 2009.igem.org
Contents |
Wet Lab
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.
