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 the two primers (0.22 (= 2/9) pmol/ul each) 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 the 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

Note:

We routinely use A18 primer and pTZrev primer shown below for colony PCR. The nucleotide sequence of the figure below is from pUC118. The multiple cloning site sequence is shown in italics.