Question

1. Design your forward and reverse primers, following the guidelines.
   1. Primer length 15-30 nucleotides

Answer 1

F.P-5'-ATTACGCCAAGCTTGGTACCG-3'

R.P-5'-ACAGCAGGGACAGGAGAAGC-3'

F.P= Number of A=5 , T=5, G=5 , C= 6

Tm of F.P= 4 * (5+6) + 2 * (5+5)= 44 + 20 = 64 C

R.P= Number of A=8 , T=0, G=8 , C= 4

Tm of R.P= 4 * (8+4) + 2 * (8+0)= 48 + 16 = 64 C

F.P Primer length= 21 nts

R.P Primer length= 20 nts

GC content in F.P= 52%

GC content in R.P= 61%

Thermal cycling for PCR

Initial denaturation= 98 C = 3min

Denaturation= 98 C= 40sec (30X)

Annealing= 59 C = 30sec (30X)

Extension= 72 C= 45sec (30X)

Final extension= 72 C = 10min

Hold= 4 C = infinite time

Denaturation is the process of making dsDNA to single stranded, so we require high temprature that breakdown hydrogen bonds but not phosphodiester bonds. So normally we keep this temperature between 95C to 99 C.

Extension temprature depends on the optimum temparature of DNA polymerase enzyme. For e.g Taq polymerase have optimum activity at 72 C so extension tempratue we will keep 72 C.

Annealing temprature we always keep 5C less than Tm of the primer to get anneales at target site.

After using above set of primer, you will get 301 bp of amplicon of above given sequence which you can visualize on the gel.