Team:Groningen/Template/MODULE/Notebook/toolbox/week5

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4 - 10 August

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Because the PCR products of NisA, PNisI and sfGFP(Bs) were lost during
the purification and restriction in the week of 28 July - 3 August, the
PCR was repeated for this genes, together with the genes that could not
be amplified yet. This time a PCR was used that did not lower in 
temperature, like the touchdown PCR, but that increased in temperature 
with each step. This way it was hoped to get over the huge gap between
the annealing temperature of the primer in the first cycle and the
annealing temperature of the primer when the flap of the primer can also 
anneal to the first PCR products. The temperature was set to increase from
40 &deg;C to 60 &deg;C in 20 cycles. Then, an additional 20 cycles were
done at 65 &deg;C. The PCR was performed under standard conditions as
was done before, together with a series of PCR that contained GC buffer
(supplied with the Phusion DNA polymerase by Thermo Scientific), and
a series of PCR with GC buffer and 1.5% DMSO. This time, with the help
of Lisa, the PCR was finally successful for all genes, see figure 3. 							
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