I am trying to design a set of primers with EcoRI ends for my gene of interest. I want to PCR the gene out of a plasmid and ligate it to 2 other different plamids with the EcoRI ends but I am a complete novice in molecular biology. Can anyone verify that what I have so far is right?
Fwd Primer: 5' TAAGCAGAATTCATGXXXXXXXXXXXXXXX 3'
Rvs Primer: 5' TGCTTAGAATTCTCAXXXXXXXXXXXXXXX 3'
I will do the PCR with a high fidelity enzyme, purify the pcr product and sequence it to make sure it is right.
One more question. I purchased a plasmid, grew it out in Ecoli, purified it and sequenced it. When I align it/blast it I get >90% identity but there are 2-3 Ns and 2 dashes within my sequence. Should I worry about the Ns and the dashes? How can I make sure the frame has not shifted and created a premature stop codon (the sequence is about 1kb and my gene is 1.4kb)?