Will this primer work? - (Dec/06/2006 )
I have designed following primers for amplifying a gene of interest which is ~981bp long using human cDNA as template. The sequence is got from a published paper(JBMR) except for few changes (RE etc). When I analyse the oligos using a software following is the result:
Forward: Total length: 30bp, Tm: 64C, GC Content: 53% (With restriction site)
Except RE Site: 20bp, 48C, 40%
Reverse: Total length: 31bp, Tm: 66C, GC Content: 55% (With restriction site)
Except RE Site: 21bp. 50C, 43%
Does they look fine? With respect to Tm, GC% and length do you believe these primers will work?
I'd be nervous about the low Tm of the region annealing to your template. In my experience, few PCR reactions work with annealing lower than 50C. Is it possible to make the binding region longer to increase Tm of that region? On the other hand, a 20 bp primer with 40% GC sounds like it should have a Tm above 48C, so perhaps your calculation is pessimistic.
i have different ranges of temperatures and bad potential structures. all of them worked just fine. however the lowest Tm i tried was 50C. am not sure about 48C.
i'm going in same direction than kathy and phage434.
It would e better to enhance the binding region of your virtual "short" primer (i mean anneal without RE site). Having a higher anneal temp for your final long primer will probably not be negative for the pcr but such a low tm will give non specific i think, especially from cDNA, wont it?
Does Tm of the total primer with RE site suffice? I mean it is 64 C & 66C... and is having a single RE (in my case both BamHI) has some disadvantageous?
This raises a good question, when you are going to add a cut site, do you include the nucleotides that create the cut site as part of your TM?
i don't. I only calculate the tm from the initial template binding seqence.
As the PCR reaction proceeds... and the restriction site becomes part of the binding sequence... the tm of the primer increases... but if the annealing temperature is kept at the initial tm.. you get a touch down PCR like effect.
Don't forget to check out NEB's site about cleavage at the ends of DNA fragments -- you might need to add nucleotides to the ends if your cleavage sites are at the ends.
While the Tm of the total primer is ~64C, only the template-specific part will do anything useful for the first few cycles of the reaction. You should aim to get that part up to 60C or so.
Having only 1 RE site does have its drawbacks, especially when it comes to cloning, but I don't think it will affect your PCR to any great extent. Because the primers have a bit of complementary sequence, they will interact, but at the temperatures you use for PCR, this effect is abolished. Can you change one of the sites to something else, or are you limited to using Bam (you couldn't use Eco RI, for example)?
Good luck and happy amplifying!