Hi there,
I have been trying to optimize primers on qPCR. Their Tm is 53.7 and 54.8. I am using FastStart Essential DNA Green Master (Roche) which has the following qPCR protocol:
1. Pre-incubation: 95'C 600sec,
2. 3-step amplification: 95'C 10 sec, 53'C - 57'C gradient 15 sec (primer dependent), and 72'C for 15 sec.
3. Melting (for melting curve)
4. Cooling
I used the same primer set from a publication, but they used biorad SYBR green mix, which uses 60'C as annealing and extension time.
I've tried the following temperature: 53, 54, 55, 56 and 57 but the highest PCR efficiency I could obtain was 85%. Also, my first Cq starts very late (Cq = 30). I also tried raising cDNA conc. but this didn't help.
(My reference gene which has Tm of 56.7 and 57.3 always produce 95% PCR efficiency at 55 and 56'C annealing temp. Cq starts usually at 25 or 26.)
Should I try lowering the Tm? I worry that the primer once annealed, would fall off as the temp gets raised up to 72'C.
Dear Min, could you calculate Tm of the primers from the link below? Take the lowest value as a Ta directly.
http://www6.appliedbiosystems.com/support/techtools/calc/
Hi. You could try to increase the primers conc. Usually 1 uM is considered enough for 60 cycles PCR. Other than that, you may want to be aware that melting temperature of the DNA depends on salt concentration, Mg2+ concentration, and also possible secondary structures formed. If I were you, I would take a look at both brand of master mix's product sheet to compare their content.
Another option is to use this technique called Touch-down PCR. It is actually just a modification of your cycling program. Let say your calculated Tm is 53.7 and 54.8oC. You can try by starting the annealing cycle at 60oC (usually starts few degrees higher from the calculated Tm). Then, start decreasing the annealing temperature by 0.5oC for each subsequent cycle until, say 50oC. The rest of the cycles use 50oC as annealing temperature. This touch down is used when we are about to try new primers and we are not too sure the optimal annealing temperature but we want to save our time.
Imagine if your actual primers' Tm are 53 and 55oC (hypothetical value for explanation sake). At the first few cycles (60 to 55oC) the primers wouldn't anneal. At 55oC, one of the primer start to anneal and the amplification begins. Another primer would anneal at 53oC and its amplification also begin from that cycle onwards. Now by the time the PCR reach a cycle where the anneal temperature is 50oC, the amplicons produced at earlier cycle would out-compete the potential primer dimer or other non-specific binding, so we don't have to worry for the non-specific binding and yet, we somehow already incorporated a Tm-optimization step in our PCR setting. This works good for those whose thermal cycler does not have the "gradient" function to do Tm optimization.
Good luck.
Dear Min, my suggestion to take Ta as 60 C and extend amplification step 72 C to 20 sec from 15 sec.
Best regards.
How long is your amplicon? Depending on polymerase speed you may need more than 15 seconds.
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