IGEM:Groningen/Notebook/iGEM 2011/2011/06/07

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07-06-11



PCR hyBB with taq DNA polymerase. Testing strain E.coli TG1 and DH5alpha.
for E.coli TG1: isolate DNA with magnetic beads of the 7th floor.
Do PCR with the isolated DNA (concentration is very low, one co worker on the 7th floor told me to use 10 to 12 microliter of the
DNA and then it should work! (at least it always worked for her))

Taq 10× buffer: 5μl
dNTPs: 1μl
MgCl2: 3μl
taq 5u/μl: 0.25μl
Forward primer 10mM: 1μl
Reverseprimer 10mM: 1μl
template: 1μl, but 12 μl for TG1 sample!
MQ: 38.75μl (but for sample TG1 use 26.75μl MQ)
PCR conditions:
denaturation: 10min
Cycle 35×
denaturtaion: 30s
annealing: 30s (gradient for 60, 65 and 70 degrees)
extension: 1min
Final extension: 10min
Store infinite at 4 degrees
Analyse them on a 1% agarosegel
PhybB has correct size!
Can use it for cloning, since taq polymerase makes 1 mistake per 1000bp according to Martijn, and PhybB plus prefix and suffix
is around 450bp.

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