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EPF-Lausanne/13 August 2008
From 2008.igem.org
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Molecular biology
Yesterday's cell cultures and mini-prep worked out fine.
F1610/E1010
We do the gel of F1610 and E1010 from the old mini-preps that we digested with SpeI and EcoRI all night. E1010 is great but F1610 doesn't work.
We don't know where the mistake comes from.
We want to be sure that the part and the enzymes are fine, so we digest F1610 and E1010 from the new mini-preps we did today, doing two experiences using both combination of enzymes: EcoRI/SpeI and PstI/XbaI.
We also do a PCR, this way we don't use restriction enzymes. We do the PCR with Biobrick primers (Prefix and suffix) for E1010, F1610, F1610/E1010(presumably assembled) and we do a positive control using DNA and Primers Bart gave us.
Finally we run a gel with :
F1610 digest by EcoRI and SpeI
E1010 digest by EcoRI and SpeI
F1610 digest by PstI and XbaI
E1010 digest by PstI and XbaI
F1610 from PCR
E1010 from PCR
Results
Restriction enzymes seem to be working fine!
E1010/F1610 from PCR (2 times from different colonies)
Positiv control from PCR
E1010 (nothing, straight from mini-prep)
F1610 (nothing, straight from mini-prep)
I1466/I14033
The gel purification doesn't work. We don't see the insert, I1433. We thus decide to focus on E1010 and F1610.
R../B0034
We focus on E1010 and F1610.
