I have primer set that has annealing temp. 60-61degree centigrade and GC content 55-60%. First step always gives good band the second step of touch down PCR gives non consistent bands under same conditions. I am trying this since last six months. what should I change ?
Hi
I guess you are annealing at 63°C? I'm not sure if I understand you right, what do you mean with first and second step? Do you mean as first step the touch down? (so probably the steps from 71 to 64)? and as second step the other steps at 63°C? Are you using overhanging primers?->and when, did you checked the temp for the complete primer binding, just as they do probably when enough material is created during the touch down? Did you checked your elongation times?
For me it's sounds like that your primers creating only good bands during the touch down, so increase the annealing temp for the non touch down steps
i do not understand the 2 steps . Are you amplifying the same product twice?. If you already have a good product .If your amplification works with touch down pcr then just use this product
What is your temp range that you are using for PCR? Sounds like you are getting good bands at the "first step" or lower temp range rather than the "second step" or higher temp. Have you tried normal PCR conditions with the annealing temp set at the lower range of temps used for step down PCR?
Hi Lipsy- Like the others, I am not certain that I know what you mean by first and second steps. However, assuming the "first step" is the series of progressively lower annealing temperatures, then I agree with the answer Michael Pichler gave. Also, I can recommend you try a range of [Mg2+] as well as temperature. If your thermal cycler will do temperature gradients, try running a gradient of temp. in the x dimension and [Mg2+] in the y dimension. With optimization of both parameters you may not even need to use a touch-down program. Good luck.
My first step is the regular PCR with annealing at 500C while second step is touch down PCR. for that also I tried from 560C -650C. I couldn't get the distinguished clear cut bands with that.
I would suggest you try it without initial PCR at 50C. The low stringency at the annealing step will generate lots of potentially improper targets which then contain the actual primer sequences (inserted during the intiial PCR). Instead, simply start at 65C and drop in 0.5C steps for 10 cycles and see how that changes things. I still recommend optimizing [Mg2+] as well.
The only concern is I am using the PCR product of first step as a template. if I don't do the first step what should be my template?
Ok, rereading your question I am now thoroughly confused. If your initial PCR is giving you the proper band, what is the purpose of the second touchdown step? If the first step is NOT giving you the proper band, then I suggest skipping it and going straight to touchdown. After all, touchdown is PCR as well, and will amplify (with optimization) anything regular PCR will amplify. Please correct me if I am misunderstanding the situation.
I am using the Nested Primers. where first step amplifies the region and second step finds the mutation. first step amplifies the region but second step doesn't give me the consistent band of products.
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