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how to make this reaction?


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#1 molbio1234

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Posted 20 June 2014 - 03:06 AM

I have some samples that have not gone through PCR yet. I need to know the ideal PCR cycle because I am targeting a yield of a certain concentration and quality of sample. I don't have enough sample volume to test PCR at different cycles. I use SYBR to measure everything (qPCR, Qubit). I have a qPCR kit with SYBR green mastermix and DNA standards. How do I use this kit to set up qPCR and determine the number of cycles and quantity at that cycle? For example, I want the qPCR to show that if I performed PCR on my samples, then at 14 cycles, I will get 500nM of product. Now when I do perform PCR on the samples at 14 cycles, I will indeed get 500nM or around there.

 

Does anyone have a protocol to set up something like this? I have qPCR SYBR DNA standards, SYBR green qPCR mastermix, and qPCR primers needed to qPCR amplify my samples.



#2 bob1

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Posted 20 June 2014 - 01:48 PM

There's no way to tell this for sure without testing it on the samples themselves.  The closest you could come is to clone the gene of interest (GOI) into a plasmid and use that to test.  However, the problem with that is that the plasmid is unlikely to have all the other junk that you would commonly find in a RNA/cDNA prep that could inhibit the reaction.  Also to work out the amount produced, you would need to know the amplicon size, the starting abundance of the GOI target, and the efficiency of the reaction - two of which you can only determine by doing the PCR!

 

Note that if you go down this route, you should clone all or most of the GOI, not just the bit between where the primers bind.






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