Science Protocols.org
Step by step methodologies

 
 

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.