Colony PCR

Colony PCR This protocol is designed to quickly screen for plasmid inserts directly from E. coli colonies. The plasmid should be high copy number such as pUC18 pUC 19, or pBluescript, etc. Even though blue/white screening can be used to determine if inserts are present, this technique can be used to determine insert size and/or orientation in the vector. Alternately, the presence of an insert and its size can be determined by growing each colony in liquid, the plasmid purified by a boiling or alkaline preparation protocol, digestion of the plasmid with restriction enzyme(s) that excises the insert, followed by separation by agarose gel electrophoresis. Typical colony PCR reaction Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 50 µl in each PCR tube (also on ice). 38 µl sterile distilled water 5 µl 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.0), 1.0% Triton X 100) 3 µl 25 mM MgCl2 1 µl 10 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP) 1 µl 20 µM forward primer 1 µl 20 µl reverse primer 0.2-1 µl Taq polymerase 50 µl total volume To each cold PCR tube containing the PCR reaction, add a small amount of colony. To do this, use a fine yellow pipette tip attached to a pipetter (set at 30 µl to avoid addition of air into the PCR reaction) and pipette up and down to mix. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields. PCR conditions 1 cycle 30-40 cycles 1 cycle 5 min at 95°C 1 min at 95°C 1.5 min at 54°C 1 min at 72°C 5 min at 72°C What happens? -initial cell breakage and DNA denaturation -DNA denatures into single strands -primers anneal to ssDNA template (temp depends on primers) -primes are extended from 3'-end by Taq(1 min/kb) -final extension to make sure all products are full length (72°C is optimal for Taq polymerase)