2 replies to this topic

[why]

I am trying to develop a PCR screening method for my samples, where some of the samples will yield 1-2 bands of certain size (~250 bp to 1.5 kb) and others will not give any band. Therefore, I want to include an internal amplification control to indicate that those PCR are working properly. I need some advice on the choice of a suitable internal control.

• Internal control size - Should I use one which is ~300 bp or ~1 kb?
  
• Internal control copy number - If the internal control is high copy number per genome, will it mask my target?

Thank you.

[bob1]

Shorter products are usually easier to amplify so I would probably go for the 300 bp.  However, multiplexing PCR like this can be very tricky as differences in primer melting temperatures between the amplicons can lead to non-specific products quite easily.

You would want something that has quite low copy-number, but you know amplifies well - plasmids are often good for this sort of thing.

Having said the above, the presence of a control band only in an individual tube doesn't necessarly indicate that the PCR didn't work on the sample, it merely indicates that the internal control primers are working.  You will probably be better off having positive and negative controls for each primer set that are run separate to the samples - so that you can say that the PCRs are working, but this sample didn't amplify for some reason.  You can then go on to do things like dilute the sample to see if there are inhibitors.

You might also want to think about having you assay set up so that amplifying a product of a certain size indicates presence, but a certain different size product indicates absence of the gene.

[why]

Dear Bob,

Thanks for the advice.