Internal Control

[slmetzger]

I need to genotype several different transgenic mice, and I found that my PCRs are unreliable. I would like to include an internal control for each reaction to ensure that my PCR is working properly and that the absence of the gene in my gel truly means that the mouse is negative for it. Can I use something like GAPDH or B-actin, similar to RT-PCR?

Thanks,
Shana

[Warthaug]

Any house-keeping gene should work - GAPDH being the usual subject as it's expression is fairly consistent across tissues. Actin would work as a positive control, so long as you are not trying to use it as an internal standard (for quantification), as its expression varies across cell types, during the cell cycle, and potentially across individual animals. So its a good measure of worked/failed for your PCR, but not the best choice if you want to measure things relative to its expression level.

If all you want is a simple wroked/didn't work readout, make sure your design your actin/gapdh/etc/ primers to give a very different sized product from your gene of interest. This way you can run both sets of primers in the same reaction, and have a clear delineation between your products.

Bryan

[slmetzger]

Great, thanks for the advice!

Shana