Given the sequence: 5’-ACTGACTATGTAGAA………GGCCCTAAGGGCCAA-3’ (1)
You wish to do PCR of this dsDNA to add “overhangs” on the ends of it. On the 5’ end of you will put the restriction site for NdeI enzyme (5’-CATATG). On the 3’ end you will put the restriction site for MspI (5’-CCGG).
Write the sequences for the “forward” and “reverse” primers to do this. Each primer should be 16-18 nt, with 12 nt for hybridization and 4-6 nt for “overhang.”
I am a little confused as to how to write the forward and reverse primers for the sequence. From videos I have seen that I should write the complementary strand of my giving strand. Which would be:
3' -TGACTGATACATCTT........CCGGGATTCCCGGTT-5' (2)
Then from sequence (1) I should get the reverse primer and from sequence (2) the forward primer. However, this doesn't allow me to add both restriction enzymes because both primers would be from 5' to 3'.
Or should I simply take the given sequence and write the primers only from that? So that the primers would be:
Forward primer: 3’-GGCCTGACTGATACAT
Reverse primer: GGATTCCCGGTTGTATAC - 5’
where the bold nts are the restriction sites.