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
* Usually, all solutions except template are premixed in a microfuge tube. They are dispensed into the PCR tubes containing 1μl of the template solution. For routine use, I make 1:1:7 mixture of 10x buffer, dNTP mix, and water containing 0.22 (= 2/9) pmol/ul each of primer and store it at -20℃. Taq DNA polymerase is added to the required amounts of the mixture just before use, and 9-microliter aliquots are dispensed into PCR tubes containing 1 microliter of template solution.
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
