TLS Online TPP Program

#Id: 2528

#Unit 13. Methods in Biology

The duration of annealing step is usually 1 min during the first as well as the subsequent cycles of PCR. 

TLS Online TPP Program

#Id: 2529

#Unit 13. Methods in Biology

The duration of primer extension is usually 2 min at 72°C

TLS Online TPP Program

#Id: 2530

#Unit 13. Methods in Biology

The specificity of the PCR depends crucially upon the primers. The following factors are important in choosing effective primers

Primers should be 17 to 30 nucleotides in length

A GC content of about 50% is ideal. For primers with a low GC content, it is desirable to choose a long primer to avoid a low melting

 temperature.

Sequences with long runs (i.e., more than three or four) of a single nucleotide should be avoided.

TLS Online TPP Program

#Id: 2531

#Unit 13. Methods in Biology

Primers with significant secondary structure are undesirable.

TLS Online TPP Program

#Id: 2532

#Unit 13. Methods in Biology

In PCR, there should be no complementarity between the two primers. 

TLS Online TPP Program

#Id: 2533

#Unit 13. Methods in Biology

The ‘Wallace rule’ (Lay Thein & Wallace 1986) is used to determine the appropriate stringency wash temperature: 

Tm = 4 × (number of GC base pairs) + 2 × (number of AT base pairs