I am performing qPCR and want to check the amplification efficiency of my target and housekeeping genes.
I have a plasmid with my target gene in that I think can be used as a template for this purpose but what do i use for my endogenous control beta 2 microglobulin?
thanks
Linearize the plasmids before using them as template.
Where you don't have plasmids, you can generate RT-PCR product (ideally with "outer" primers, but you can also use the qPCR primers to generate that product).
If the gene is highly expressed it may be sufficient to use the cDNA from the RT as standard.
If you are going to use plasmids and/or PCR product please be very careful to avoid contaminations. Do all prep steps in a different lab (ideally in a different building) and bring only the working dilution (containing about 1E4-1E5 copies/µl) into the qPCR lab.
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