Question

The forward and reverse primers for PCR amplification have the following sequences: Forward: 5'-CTATGAGTTACTCACATG-3', Reverse: 5'- GAAAGCATCCAGGCCCGG-3'. Based on the manually calculated melting temperatures, would you recommend using these primers together? Why or why not?

Answer 1

The Tm of forward primer according to formula

Forward: 5'-CTATGAGTTACTCACATG-3'

Tm = 2 X (A+T) + 4 X (G+C)

is 2*11+4*7=50 *C

5'- GAAAGCATCCAGGCCCGG-3'

The Tm of reverse primer according to formula

is 2*6+4*12=60*C

The difference between Tm of the primer pair is 10*C,i.e more than 5*C difference.Thus primers with Tm difference greater than 5*C is not optimal and can lead to no amplification.

Also the forward primer has GC<40%

and the reverse primer has GC>60% while the ideal GC content should be 40-60%

Thus i would not recommend these primers since they don't go with the guidlines of primer formation and may lead to no amplification.