I have a set of primers designed by IDT and the Tm for most of them is 62 but I am using Quant fast SYBR green which has an annealing temperature of 60 C and says that the primers should have the Tm below 60. In my qPCR condition, I am using 60 C for 45 seconds and for some of the primers this seems to work, however for a couple of them this does not work. My question is whether the annealing temperature should be the one recommended in the kit or if we can change it to optimize our condition?
Also, should I order primers with Tm below 60 C?
Because my samples are not enough to test for various conditions, I am afraid to lose them.
why do you need to use 60? the higher the tm the harder for the primers to bind but of course increase the specificity as well.. It is better to optimise first as always.. but in my case I always start from 55C.. most of the primers i used have Tm more than 60.. but my qPCR works well.. you can check the results by seeing the melting curve..
I would suggest you to optimize the PCR condition, becouse with kit you get some recomded condition but you no need to worry, and if your primer Tm iz more mean it increase yhe specificity.
You should start from the recommended Tm of the kit and go upto your highest Tm thus optimize the reaction. Because different polymerases have different Tm calculations. First you check whether they have recommended any specific calculation or not. Like if you are useing Thermo's phusion polymerase, they have a different calculations for Tm of your primers and you have to use that calculation for optimal results.
You didn't specify the problem with the "primers that didn't work". Didn't they amplify at all or did you find some unspecific bands? If it's the second case, try to keep Tm=60°C but increase MgCl2 concentration. Hope that was useful
I ran into this problem recently and found out that the truth is in the fine print. Almost all kits and primer design programs will say in the fine print something similar to "all calculations of primer annealing temps are estimations and ideal annealing temperatures should be determined for each individual assay". They are basically saying that their calculations are estimations or make a lot of assumptions. Don't waste a bunch of SYBR, troubleshooting in qPCR. It's too expensive. Do a regular PCR with cDNA and use a temperature gradient to find the proper annealing temp for your primers. If your PCR machine doesn't have a gradient, run several PCRs at 55, 57, 59, etc... You have the ideal temp when you get one strong band. Then, transition to doing qPCR and further troubleshoot the temperature there (you probably won't have to). Important to mention, if the kit says the maximum is 60C, don't go above that. Redesign your primers (which are cheap) if you need a temp above the maximum for the assay. Good luck!
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