PRIMER 3 PROGRAMME - (Jan/09/2007 )
im trying to use the primer 3 programme on the net to check the primers im using ,,,
the problem is that i didnt know how to use this programme ,,,can anyone help?
what do you mean by check?
Check that it matches a sequence, or check the tm and gc content?
to check primer dimers i use:
it give the length, gc content, melt temp, molecular weight, extinction coefficient, nmol/od260, and ug.od260....
to check that your primers amplify a sequence, there are some in silico PCR programs on the net available (don't have the address at hand, google should do it)... or run it in blast.
thanks for ur reply ...the problem is that i wanted toknow the suitable temp for annealing but i put the primers in there spcaes but the page doesnt resp[ond by any information !!!
to test annealing temps, i do that manually, ie you'd use a gradient PCR. if the melting tm is 60'C, then the annealing temp is probably around 55'C. so you'd just have to muck around with getting the cycle right.
yes you dont have to calculate it, do as vetticus said, or simply i go 2C below the Tm and see what happens. if you have two cyclers (our lab ) you can try different temperatures together.
i know thee role about the 5 degrees below or above the Tm ... the prroblem is that im working
on a multiplex PCR including 8 forword and 8 reverse primers and the primers have different Tm
ranging from 52 to 70 ...i tried temps at 58 57 56 55 54 53 and alll were not that good i dont know what to do
with a range from 52 to 70... i'm inclined to think that PCR will never work. perhaps you should redesign the primers so they have a more similar tm.
Tm 70 why is that so? i think more than 60 is too high.
I've done PCR with one primer 60 and another 79. (High GC content). It's alright, I set my thermocycler anneal temperature to 60 and added PCR enhancer (betaine, DMSO or "Q solution"). Search on this forum for high TM PCR.