p16 PCR

[Anonymous]

I am attempting to amplify p16 cDNA. I know my primers work and the PCR cycles are working as I have a p16 positive control which works in all my attempts. However I get very erratic results, but on average I get poor bands for my samples when I perform gel electrophoresis.
As far as I can make out I think my samples (obtained from lymphocytes) simply have very little p16 mRNA in them.
Is it sensible then to perform one PCR reaction and then use a aliquot of my PCR products from the first PCR reaction and perform a second identical one? Would this potentially show stronger bands on my gel?
Importantly I am looking for either an up regulation or down regulation between the samples i want to amplify.

Thanks

Chris

[Anonymous]

Hi Daniel,

That sounds like excellent news. Performing RT-PCR is my ultimate goal, I have access to an applied biosystems stepone plus light cycler where i hope to eventually use a sybr green dye to look at p16 mRNA. However I’m stuck at getting the the whole thing to work reliably at the moment.

I have attempted performing what i mentioned, but because I was unsure if using simply a neat sample would work I also created dilutions of 10, 100, 200, 1000 and 2000.

Will get let you know if it has worked!

Thanks

Chris

[Daniel]

Yes you can do this. It is better to do nested PCR where you use internal primers for the second round PCR.

If you want to measure expression levels of your mRNA your really need to do real-time PCR – the results from just looking at the amount of PCR product produced in the end will really on be semi-qualitative at best.

Daniel Tillett
Nucleics Support

[Author]

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