I am trying to amplify a gene in same conditions (i.e. melting temp, annealing temp and extension temp and time for all steps) on which I have previously amplified this gene. But this time I am not getting amplifcation.
Suggest any possible reasons.
Hi,
sometimes, the annealing temperature could change with a new polymerase (mix) lot. Also, you can have SNP under the primer or some deletion in the primer annealing region can be present. It is not clear whether you analysed the same DNA sample or another one.
If another, use the first succesful one as a control. If the same one, you should do gradient PCR or try to find a new annealing temperature around the first one.
Good luck
Marcela
- I agree with Marcela
- I have known many instances where switching from say Taq to pfu polymerase can change the annealing temp
- if that has been done perform an annealing gradient in which you vary the Tm in 1C intervals from to Tm-3C to Tm
- Indeed if you are now using another prep because of variation in quality and/or quantity of DNA the melting temp might not apply
- If you are using a different prep to before try dropping the annealing temp by 1-2C
Hello Akshay
To rule out all you reagents are good condition have a positive control
If thats fine, as suggested above changing the reagents(different company) may affect your annealing temp.
If your reagents are the same and if they are working perfectly (including primer, poly, buffer, dntps) try to adjust Mgcl conc (it may work out)
Even if it does not work out, do a gradient PCR which will give u a good idea of what your dealing with
And if you could mention the condition it will be useful.
is it conventional or Qpcr?
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