We are doing gene pyramiding in wheat for which we are using 5 markers for different genes. The main problem is that a primer is giving results on the first time of use but on the second time we are not getting any results or differnt bands on the gel. I am facing this problem with 3 markers we also use some other SSRs present in the lab but they are giving very good amplifications.
Tinku ji,
please make fresh working primers from stock then try again. Run a positive PCR with this to check quality of PCR constituents.
If still no amplification and "other material is perfect" then try to standardize annealing temp again. its very common in PCR not to amplify same gene with the same method.
wishes...
Some time it happens. Change the annealing temperature and also try by taking different sample of same species. Check out whether primer is in good condition or not. you can add some more Mg.
All the best..
Dear Avneesh ji thanks for your responses but we have tried every thing.
1. Primer stocks from four differnt companies.
2. DIffernt MgCl2 concentrations (1 mM-6mM)
3. Different enzyme concentrations.
4. Different Primer concentrations
5. Gradient PCR.
6. Touch down PCR
7. Different Template concentrations
Every time we get a smeer at the position of the expected size.
What else one can do...
Smear shows that your reaction is going on .....have you tried positive control with the same DNA/cDNA? if yes then try MgSO4 in place of MgCl2....i got better result with this..
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