What annealing temperature is better for my primers? - (Aug/09/2006 )
Hi, there, I have a pair of primers, both Tms are 78 degree. Does anyone know what the maximum anneanling temperature can be set? I set the temperature to 68 degree. Agrose result showed there were 3 non-specific band less than interest size. But no interest band was found.
you could try to do a 2-step, with annealing and extension combined into one step at 72; that may allow for a little better specificity
however, the lack of band at the correct size is worrisome. are you sure of your template? would you please post a pic?
Is there a particular reason why the Tm is so high? Is the sequence very GC-rich? If so, you could add some DMSO or betaine to reduce non-specific binding. Do you have restriction sites on the ends? If so, recalculate the TM without those bases, you'll get a better indication of what's going to bind to the template...
I would suggest you also think about shortening the primers a bit.
Anyone in your department got a Gradient PCR machine?
See if you can obtain the use of one. They're really good if you're unsure about annealing temps. Also make sure to do a Touchdown PCR first to check you have the right conditions.
I would suggest you also think about shortening the primers a bit.
Hi, Swanny, I recalculated the Tm without restriction sites. Tm decreased to just above 50 degree. Do you think these will make any sence? Thanks.
What formula are you using to calculate tm?
-Matt
Is there a particular reason why the Tm is so high? Is the sequence very GC-rich? If so, you could add some DMSO or betaine to reduce non-specific binding. Do you have restriction sites on the ends? If so, recalculate the TM without those bases, you'll get a better indication of what's going to bind to the template...
I would suggest you also think about shortening the primers a bit.
Hi, Swanny, I recalculated the Tm without restriction sites. Tm decreased to just above 50 degree. Do you think these will make any sence? Thanks.
That is starting to make more sense, but I think you might now have the reverse problem, sorry to say. I think that your primer sequence might not be long enough...
If you tried to do an amplification, your annealing temp would be so low that the Taq would not be functional, and by the temperature it was working (and therefore extending the primer) your primer would have fallen off.The basic rule that I learned was to have the template-specific primer Tm about 67-70C +- 3C, then add the restriction site sequence, and use an annealing temp between 60 and 68C. You might need to remake the PCR primers to take this into account.
you can also try a touch down pcr for decreasing non specificity.