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 * 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) |
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