Design two 10mer PCR Primers two primers that are each 10 nu

Design two ‘10mer’ PCR Primers (two primers that are each 10 nucleotides in length) that will prime the 5’ and 3’ ends of the following DNA sequence. But also add EcoRI restriction sites onto the ends so that, once this sequence has been amplified, you can insert it into a cloning vector.

EcoRI site:

5’-GAATTC-3’

DNA Sequence:

5’-ACTTCGACTGGCG(5000bp)GCTTACGTAGCTTAGGCT-3’

3’-TGAAGCTGACCGC(5000bp)CGAATGCATCGAATCCGA-5

Solution

DNA Sequence:

5’-ACTTCGACTGGCG(5000bp)GCTTACGTAGCTTAGGCT-3’

3’-TGAAGCTGACCGC(5000bp)CGAATGCATCGAATCCGA-5’

EcoRI site:

5’-GAATTC-3’

Adding EcoRI site in the DNA sequence we get

5’-ACTTCGACTGGCG(5000bp)GCTTACGTAGCTTAGGCTGAATTC-3’

3’-TGAAGCTGACCGC(5000bp)CGAATGCATCGAATCCGACTTAAG-5’

Forward primer: Complimentary to the 3` end of the 3`- 5` DNA sequence

Reverse primer: Complimentary to the 5` end of the 5`- 3` DNA sequence

So, the `10mer` PCR primers are:

FORWARD PRIMER 5`-ACTTCGACTG-3`

REVERSE PRIMER 3`-CCGACTTAAG-5`