Design the forward and reverse primers to the following genes. Write answers in

Question

Design the forward and reverse primers to the following genes. Write answers in the 5’>3’ direction. Primers are usually ~20 nucleotides with Tm’s around 45-60°, but only design them to be 6 nucleotides long to
reduce work. Calculate the Tm of each primer, and determine what annealing temperature you would use in PCR. You do not need to look for a start codon, as this is a random sequence of DNA.

13. 5’-GCTGTAAGAGGATTTACTACCGTCCA-3’

Forward: ______________________ Tm: _______

Reverse: ______________________ Tm: _______

Annealing temperature: ______________________

Explanation / Answer

Given DNA sequence: 5'-GCTGTAAGAGGATTTACTACCGTCCA-3'

Forward primer: 5'-GCTGTA-3'

Length: 6 ntd

GC content = 50%

Basic Tm = 18'C

Reverse primer: 5'-TGGACG-3'

Length: 6 ntd

GC content = 67%

Basic Tm = 20'C

The annealing temperature should be 2-3'C less than the Tm. It also depends upon the buffer composition used for the PCR reaction.

So, the annealing temperature = 15'C

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