15-07-11
Plates of yesterday's transformation looks better than transformations before. Self ligation plates contain 5 to 10 times less
colonies than the rest of the plates.
Colony PCR was done as followed:
Mastermix scheme:
Taq 10× buffer with NH4SO4 without MgCl2: 58.00μl
dNTPs 10mM each: 11.6μl
MgCl2: 34.8μl
Taq polymerase 5u/μl: 2.90μl
Biobrick vector forward primer 10μM: 11.6μl
Biobrick vector reverse primer 10μM: 11.6μl
MilliQ water: 449.50μl
PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle (33×)
Denaturation: 94°C for 30s.
Annealing: 60°C for 30s.
Extension: 72°C for 2 min.
Final extension: 72°C for 10 min.
Store at 4°C infinite
PCR products were analysed on a 1% agarose gel with TBE.
Results: PBAD-cI-LVA-DT colony 1 seems to be right (correct size on gel (2kb) )
PhybB-cI-LVA-DT no right transformants.
PBAD-RBS-GFP-DT colony 4 looks alright, but is difficult to see because the gel is not very good, so just try along with the
pBAD-cI-LVA colony 1 for plasmid prep, glycerol
stock and sample for sequencing
Transformation again with samples of yesterday (ligation mixtures were stored in the frige).
Same protocol as yesterday.
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