PCR from bacterial colonies
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This procedure is used to analyze inserts in pUC-derived plasimids.
1. Suspend E. coli colonies harbouring plasmids in 50 microliters of TE.
2. Incubate for 5 min at 95 degrees or in boiling water.
3. Mix the following solutions.
Template solution prepared as above 1 μl
10x Taq buffer 1 μl
dNTP mix (2.0-2.5mM each of 4dNTP) 1 μl
forward primer 2 pmol
reverse primer 2 pmol
H2O up to 10 μl
Taq DNA polymerase 0.1 unit
4. Perform PCR following standard procedure. Twenty five cycles are enough for analyzing pUC-derived plasmids.
Solutions
TE
10 mM Tris-Cl (pH 8.0)
0.2 mM EDTA
