I designed primers for specific exons, the exons length are 3.6 kb, and 1.9 kb respectively. While standardizing the PCR with different temperature I did not find any band on gel. So again I repeated with different temp. by adding DMSO. This time non specific bands was found.
So let me help to reduce the non specific band formation
Thanks in advance
Dear Yedukondalu
For optimization of pcr and determination of suitable annealing temperature, you should use of gradient runs. For reduce of nonspecific bands, you should use of low concentration of MgCl2. As well as, you can use of Hot start pcr technique (for prevent contamination of your materials).
Best, Roshdi
Another nice method to reduce non-specific bands is the touchdown pcr. There you start with "too high" annealing temperatures and lower them from cycle to cycle. The rationale behind this is to pre-amplify very specific products (high Ta = high specificity, low yield) and later shifting to normal Ta (lower specificity, high yield). I usually do 5 cycles (62, 61, 60, 59, 58°C) and then 30 cycles with 57 °C if that´s your optimal annealing temperature. Gradient is of course also an option, but it is quite a waste of material and enzymes. I only use it as a last resort.
With DMSO you have to be careful not to use too much. Start as low as possible and keep in mind you have to reduce your annealing temperature by 0.6°C per percent of DMSO used. Don´t exceed 10% in total since it also facilitates polymerase errors sometimes.
Hope this helps! Good luck!
Flo
additionally to the above answers try adding a final concentration of 1M betaine in the reaction in case the template does not melt easily. Also run a control primer set to check that the dna does not have inhibitors and is present in good amounts so that you can be sure that amplification can take place. I think gradient pcr is an excellent way of getting the correct annealing temperature
design another set of primers and check their properties like forming hairpin and primer dimer by using softwares like primer3. because main reason behind nonspecificity is the primer sequence.
Primer design is the most critical issue, but during optimization you will usally see non-specific amplification. What I do, in this order (little added to the above mentioned suggestions):
1) Annealing temperature gradient.
2) Lower MgCl2 concentrations
3) Hot-Start Taq
4) DMSO
5) Betaine
6) DMSO+Betaine (sometimes works really well).
7) Touchdown PCR.
If the design is not optimized at this point, try a new primer design.
I would not use DMSO or betaine for Real-Time PCR, though.
Good luck!!
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