McClean:Touchdown PCR
Background
Use this PCR to avoid primers amplifying nonspecific sequences. The procedure is based off of 2 principles. First, the specificity of the products is based off the annealing temperature at which the reaction occurs. That is, the lower the temperature, the lower the specificity. Secondly, the melting temperature of the primers sets an upper limit to the annealing temperature. By keeping the temperature very close to the melting temperature of the primers initially, the most specific products will be amplified first and thus will not be "swamped out" by the nonspecific products as the temperature of the reaction is lowered. Moreover the initial amplification causes there to be more template of the specific sequence available for amplification also helping to out compete nonspecific products.
Procedure
Reaction mixture
1X Reaction (Total Volume = 50 μL)
10x HotMaster Taq buffer with Mg2+ 5μL 10 mM dNTP mix 1μL 10 uM Primer 1 0.5μL 10 uM Primer 1 0.5μL 20ng/μL Template DNA 2μL HotMaster Taq DNA polymerase 0.5μL Sterile Water 37.5μL DMSO 2.5μL
PCR conditions
Run on PCR machine under HotStartPCR program
- 95°C for 4 min
- 94°C for 1 min
- 75°C for 1 min; -1°C each cycle
- 68°C for 2 min
- Repeat 2-4 30 times.
- 94°C 30 sec
- 55°C 30 sec
- 72°C 1 min
- Repeat 6-8 30 times
- 75°C 5 min
- 4°C --end
