Typical colony PCR reaction
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
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
PCR conditions
• 1 cycle
· 5 min at 95°C => Initial cell breakage and DNA denaturation
· 1 min at 95°C => DNA denatures into single strands
• 30-40 cycles
· 1.5 min at 54°C => Primers anneal to ssDNA template (temp depends on primers)
· 1 min at 72°C => Primes are extended from 3'-end by Taq(1 min/kb)
• 1 cycle
· 5 min at 72°C => Final extension to make sure all products are full length (72°C is optimal for Taq polymerase)
