Standard PCR with Taq DNA Polymerase
Updated 5/17/2014 08:01 PM
Introduction
Materials
- Reaction Mixture (50µL)
- 5µL 10X Taq buffer
- 1µL 10mM dNTPs
- 1µL 10µM forward primer
- 1µL 10µM reverse primer
- 10pg - 1µg template DNA
- 1.25U Taq DNA polymerase
- Water, nuclease-free
- PCR Tubes
- Thermocycler
Procedure
- After thawing, gently vortex and centrifuge all solutions.
- Assemble all reaction components in PCR tubes on ice.
- To prepare several reactions at once, create a master mix with all components except the template DNA. Then combine this with the DNA in each PCR tube.
- Gently mix the reaction, using a quick spin to collect liquid if necessary. Overlay the mixture with 25µL of mineral oil when using a thermocycler without a lid.
- Quickly transfer PCR tubes to the thermocycler pre-heated to the 95°C denaturation temperature. The recommended thermocycler conditions are as follows:
Initial denaturation: 95°C, 1 min (longer for GC-rich sequences)
25-40 cycles of denaturation (95°C, 30 secs), annealing (primer Tm, 45-68°C, 30 secs), extension (68°C, 1kb/min or ~5 mins)
Final extension: 68°C, 5 min
Hold: 4°C
PCR (polymerase chain reaction) is a technique used to amplify specific regions of DNA by several orders of magnitude. It makes uses of primers (short DNA fragments) that are complementary to the desired DNA region and a DNA polymerase. Through repeated cycles of heating and cooling, DNA generated from the original template is eventually used as a template itself, resulting in exponential amplification.