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 10x Taq buffer dNTP mix (2.0-2.5mM each of 4dNTP) forward primer reverse primer H2O Taq DNA polymerase

1 ul 1 ul 1 ul 2 pmol 2 pmol up to 10 ul 0.1 unit

* Usually, all solutions except template are premixed in a microfuge tube. They are dispensed into the PCR tubes containing 1ul 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 C. 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