IGEM:Groningen/Notebook/iGEM 2011/2011/06/15

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15-6-11


Plasmid prep of overnight cultures
ND1000 nanodrop measurements: DNA concentration is between 30 and 50 ng/μl

Searching for protocols for PCR with taq and pfu polymerase
PCR of promotors PlasB, PBADaraC, PhybB

PCR with taq:
10× taq buffer: 5μl
10mM dNTPs: 1μl
BB forward primer10μM: 2.5μl
BB reverse primer10μM: 2.5μl
Taq DNA polymerase: 0.5μl
MgCl2 25mM: 4μl
MQ water: 33.5μl

PCR with pfu:
10× pfu buffer with MgSO4: 5μl
10mM dNTPs: 1μl
BB forward primer10μM: 1μl
BB reverse primer10μM: 1μl
Pfu DNA polymerase: 1μl
MQ water: 40μl

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 2,5 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C

Analyse on a 1% TBE agarose gel.
Clean up DNA with High Pure PCR Purification Kit

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