1 I got both outer and inner band for wild type on temperature but no band for mutation now i am getting no any band on the same conditions on which already got band while optimization.
2. if I don't have any positive sample of mutation then how can I confirm about primer specific for mutation is working or not!
You really do need a positive control.Is it possible to get samples probably positive that you cab screen (even by sequencing if necessary). If not you could test the temperature for annealing of the mut primer by buying a long oligo with the mutant primer sequence at one end and a lower annealing primer at the other .Then run a temperature gradient which should give you an annealing range for the mutant primer
The answer is very helpful .
Can you please explain what can I do if I am getting only band of outer and i have run gradient temprature pcr,too many times by changing as well.
I see from your question that you were getting both outer and one inner amplimers. If you are not getting the inner any more then it suggests that your inner primer has degraded. This may be because it was dissolved in water (which is acidic) not TE which has alkaline buffering and EDTA to inacxtivate nucleases. I would re order the inner primer or take another alliqout from its frozen stock and hopefully with good quality primer the normal band will reappear. The mutant is more of a problem because it never has worked so we do not know if it is in good shape. I would check that the mutant primer is designed correctly to be able to give an amplimer with the outer primer. Again go back to your original primer stock and use a new aliquot and amplify using too low a temperature (10c below the calculated annealing temperature. If this fails to give a band then reorder the mutant primer and dissolve in TE and store some aliquots in small tubed to avoid continual re freezing.
Once you can get amplimers from both primer sets you can screen some samples and hope to find a sample which fails to amplify the normal but does amplify the mutant in a search for a homozygote positive control which can be used to analyse the highest annealing temperature for the mutant primer set.
Do your gel results have any small primer dimer smear at the low size region of the gel with any of your samples or no template control samples?
In that case you should try running samples with half, quarter and eighth the amount of all primers. If you have it then use a hot start polymerase to minimise P-D. The PD band may look weak but it is very small so does not trap much ETBR so looks faint but actually contains a lot of primer and sometimes so much primer is removed as PD that the pcr fails. It would be intersting tosee if both outer and mutant primer only and also both outer and normal only produce a pd. If only the mix with mutant primer forms a PD then this may be part of the problem.Try hot start taq ( but absolutely not a proof reading polymerase) first.
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