[relaswar]

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 !

[TonyBrooks]

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 !

I would agree wholeheartedly here. It's worth the effort optimising the PCR before you begin as it saves all the troubleshooting headache further down the line.
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.

[pmiguel]

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