Primer design with EcoRI ends - (Mar/08/2015 )

Hello,

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)?

Thanks

I use to put only 3 bases which works fine, so yours should work even better as per NEB.

about your second question, check out the chromatogram where these N's and dashes are, i assume these errored bases might be close or too far from the sequencing primer. As the good sequencing coverage starts approx after 50-75 bp from the sequencing primer used and you can trust the call until 600-700 bp from there.  if you want to sequence your whole 1.4 kb gene you need atleast two sequencing primers to walk around the entire gene.

I don't know what your next step is, but usually cloning with two different enzymes is easier (and directional). You should, of course, determine if there are EcoRI sites within your gene. As GNANA says, you can use additional sequencing primers to assure that your sequence is correct. Errors usually occur before about 120 bp and after about 700 bp from the primer. Many of these can be resolved by careful inspection of the electropherogram.