McClean:Touchdown PCR

From OpenWetWare

Jump to: navigation, search

Contents

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

  1. 95°C for 4 min
  2. 94°C for 1 min
  3. 75°C for 1 min; -1°C each cycle
  4. 68°C for 2 min
  5. Repeat 2-4 30 times.
  6. 94°C 30 sec
  7. 55°C 30 sec
  8. 72°C 1 min
  9. Repeat 6-8 30 times
  10. 75°C 5 min
  11. 4°C --end
Personal tools