Touchdown
PCR Protocol
Method to
perform Touchdown PCR. Generally results in specific
amplification of desired PCR product without optimizing the
PCR protocol.
1. Determine the melting temperature (Tm) of the
primers (for the purpose of this protocol we will use
60C).
2. Set up a PCR machine with touchdown program
(5 degrees higher than the Tm, that reduces ½ a degree per
cycle until it reaches 5 degrees lower than the Tm).
3. The example set up:
|
94
C
|
5
mins
|
1
cycle
|
|
94
C
|
30
seconds
|
20
cycles
|
|
65 C to
55 C
|
45
seconds
|
|
72
C
|
2
mins
|
|
94
C
|
30
seconds
|
20
cycles
|
|
55
C
|
45
seconds
|
|
72
C
|
2
mins
|
|
72
C
|
5
mins
|
1
cycle
|
|
4
C
|
Forever
|
(pick
up samples)
|
4. Create PCR Mastermix as normal, no need for
specialised reagents.
5. Add DNA to required volume of
mastermix.
6. Place in PCR machine and begin
protocol.
|