Optimum annealing temperature - Tm calculator (Aug/23/2007 )
Gradient thermocyclers often use a fan to blow cool air across the heating plate during the annealing step. The annealing temperature is set at a nominal temperature by the user, say 65, or 67, or whatever annealing temperature you choose. The wells nearest the fan lose the most heat, which gives them the lowest annealing temperature. Conversely, the wells furthest away from the fan are cooled the least, so they have the highest annealing temp. This way, your generate a temperature gradient of 5 to 8 degrees across the plate. The particular machine will often say what the gradient is. You run a gel of the PCR, and select which wells give the best response.
Is it critical that you have a specific temperature, or do you really just want a strong amplification? If so, you could just do a touchdown PCR, where you start with an annealing temp above the theoretical Tm, and every couple of cycles you drop the temp by 1 degree, until you are several degrees below (this could be over 20 cycles). Finally you do 10 cycles or so at the lowest annealing temp.
I have 3 different Tm readings when i use 3 different software. i designed my primers based on vectorNTI and Primer3plus. However, when i ordered my primers from Research Biolabs, they gave very different readings as well as a large gap between the forward and the reverse primers. Ihave check the primers for primer dimers as well as primer-primer interaction. They gave good results
I have been running PCR with a gradient thermocycler, i find that the results are very inconsistence. Bands are weak ( i use TBE 1x and SyberSafe, Mastermix, hot start, 1 ul template and 2ul primers ). I have tried many times with new isolated templates.
Also do the intergrity of the wash buffer used in qiagen blood and tissue DNA isolation kit affect the quaility of the template thus resulting in failure of my PCR? Will traces of PCR inhibitors found in the wash buffer cause the inhibition of PCR
Is the inconsistency due to error in annealing temperature?
I have been running PCR with a gradient thermocycler, i find that the results are very inconsistence. Bands are weak ( i use TBE 1x and SyberSafe, Mastermix, hot start, 1 ul template and 2ul primers ). I have tried many times with new isolated templates.
Also do the intergrity of the wash buffer used in qiagen blood and tissue DNA isolation kit affect the quaility of the template thus resulting in failure of my PCR? Will traces of PCR inhibitors found in the wash buffer cause the inhibition of PCR
Is the inconsistency due to error in annealing temperature?
Working backwards through the message:
Probably. Check the difference in Tm; if it's more than 5C, your efficiency drops.
Look, I have always used the oldest, cheap-and-dirty calculation for Tm - the old "2C for each C or T, 4C for each A or G". Try that calculation on your two primers, and if the Tms are too far apart, add a couple of bases to the lower Tm primer.
Traces of PCR inhibitors will inhibit, but if your extraction kit is not contaminated, there shouldn't be enough to cause a problem. You have to follow the protocols correctly to get the correct results. The chemistry is so well-established that they are almost guaranteed to work.
If you use well-balanced primers and still don't get good amplification, try titrating your MgCl2.