22-6-11
Transformation of overnight ligation
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight
Meanwhile, prepare the plates, ampicillin plates
Obtaining more PCR product:
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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