Primer with tag: Which is the real concentration? - (Jun/22/2006 )
If you have one primer (20bp) with a tag (12bp) for, let's say, adding restricion sites, how I calculate the concentration that I need in my PCR? How do I calculate the melting temperature?
I think that for the annelaing temperature you only use the primer section that join your target sequence and assume that the tag doens't affect this annealing temperature.
About the concentration and in order to use equimolar primer concentrations (forward and reverse), should I multiply the total concentration (primer+tag) by, in this case, 20/32, because that is the part which will join to your template.
you know, there was a thread not too long ago that covered all this...I couldn't find it to give you a link, so I will summarize a little:
the tag will not change your concentration. you want molar quantities; you don't care about ug...and molar quantities won't change just because the primer is longer. do you see?
for annealing temp....you are mostly correct it is best to set up your PCR in such a way as to maximize specific product, which means your program will be a little complicated. I'm going to give a 'sample' protocol; specifics will depend on your template, your product length, and your polymerase:
55 45s (for first annealing temp; where primers match gDNA)
X 5-15 cycles; I usually do 8 or10
then switch to second annealing temp, using entire length of the primer. this is because you are amplifying the product you have made in the first 10 cycles. oftentimes adding restriction sites and tags will give quite high Ta's and it's just easier to do two-step PCR like so:
X 25-30 cycles; I usually do 25, things might be depleting if you push it too much
then do your extra extension at 72 for 3 min or 5 min or whatever you like
Do you see? the protocol has like a dozen or so steps you have to program into your cycler, but you will get the maximum amount of specific product
Clear as crystal.
Very well-explained. I don't have doubts about this matter any more. And I will put into practice as soon as I can.
Thank very much (seriously).
you're wise enough to ask before you start; I had to learn that one the hard way myself
tried to get a PCR several times...was going crazy...realized I needed to initially lower the Ta to get the primers to bind to the gDNA