need anealing temp for PCR - (Mar/08/2007 )
hi i designed some primers and that primers have follwing Tm after -5
1) fw 59 and rev 59
2) 67 and 69
3) 73 and 69
4) 65 and 69
5) 67 and 69
6) 63 and 61
i run with different temp like 55,53 and 50 and 63 and with 35 and 40 cycles.
my pcr programme is
94 30 sec
aneaing temp 30 sec
72 1 min
4 for ever
but i am not seeing any single bands and i am seeing many un specific bands.
please suggest me how to solve this problem. any ideas and anything to get better results.
The Tm of some of the primers are pretty high, that # is the given by company that prepare the primers or is the estimate Tm? You Could try a promedy between the lower and higher the temp. that will be 66C for the temp given. Because the temp is so high I recommend that add a temp ramp (decrease about 5-10% time rate of heating/cooling) between the denat step and annealing. If the product is more that 400bp anneal for at leat 45 sec. hope this help.
thank you for reply. what is temp ramp. i didnt understand . i used 63 temp but it doesnt worked.
i calculated Tm using formula A and T *2 and G and C *4 and -5.
what you suggest
thanks in advance
A couple of things Ravuri. That method for calculating Tm can be unreliable. Use the nearest neighbour method. Go to the Finnzymes site, they have a Tm calculator using the nearest neighbour method. If you are getting non-specific bands, you can use a touchdown PCR. That's where you start with annealing at 70C for example, and each cycle after that anneals at 0.5C less, so you go to 69.5, then 69.0 and so on. All cyclers have this function. The high temperature ensures the primers will only bind to their specific targets if anything, and the decrease in temperatures allows the primers to bind to the target if they do not bind at the higher temperatures. Also check the GC% of your targets. If they are high (>55%) your reactions may require DMSO or betaine to reduce the strength of GC bonds and allow your template to be more easily denatured and your target to be isolated.
A ramp is a function of the machine were you can control the speed of temp. change. Some times when the temp change to fast the primers don't stay in the strand (like they fall of") don't know why happen but have seen it. Yo can make the temp to change slower than the default. Other thing that you can do is check is you machine have a temp gradient
this mean that each well have a different temp. so you make several samples the one that have the best band, that's the temp that you need. You can use the formula to estimate a tm, but there is several things that make the tm different to expected. Check first the document that came with the primer and check whats the tm given by company.
aside from the above comment,
If the tm quoted are correct, I would suggest using additive like DMSO (1%-5%), betanine (0.1M -0.5M), to help lower the tm. Glycerol and BSA, can be added they might help improve stability of the polymerase.
I would also suggest that you limit the number of cycles to no more the 35. Above 30 cycles, the PCR reaction plateaus.
You can also reduced the melting time from 30 sec to 15sec or even 10sec, depending on the size of your product, which from the looks of the extention time, isn't very big.
Assuming the tm are really this high, reducing the annealing time to 10sec can help.
I would suggest using additive like DMSO (1%-5%), betanine (0.1M -0.5M), to help lower the tm. Glycerol and BSA, can be added they might help improve stability of the polymerase.
what % of glycerol?
Again it is 1% to 5% Glycerol.
PS: In really desperate conditions the betanine concentration can be increased upto 2M.
I go with killerkoz17. Touchdown is great. And to make sure, I'd add a bit of betaine (1%) or DMSO (0.1 M) as pernesblue suggests.
It sounds like you are doing single target reactions, yes? If you're doing multiplex, check each of the individual reactions to see if one in particular is bad.