I wonder if rigorous optimisation of PCR assays will alleviate some of the problems related to the production of non-specific products? Also, this might yield higher proportion of desired product which will preferentially overtake annealing of smaller/non-specific products in emPCR. Just a thought !
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Originally posted by relaswar View PostI wonder if rigorous optimisation of PCR assays will alleviate some of the problems related to the production of non-specific products? Also, this might yield higher proportion of desired product which will preferentially overtake annealing of smaller/non-specific products in emPCR. Just a thought !
Make sure the input DNA is of sufficient quantity and quality.
Avoid GC rich primer sequences.
Use a good quality hotstart enzyme.
Increase Tm
Add 3% DMSO
Double AmPure purify / gel-cut
etc
Remember: rubbish in = rubbish out.
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I think it might. If optimization were done to eliminate any primer dimers then I think the majority of the issues go away. But you would want to assay for primer dimers with little or no clean-up of the reaction. Maybe on a 1.5-2% agarose gel. Just load 10% of the reaction on the gel, run and take a look.
If you don't see any primer dimers, then it is unlikely there will be any single-stranded primer dimers annealed to the adapters of your main products.
--
Phillip
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