Any clue to this PCR question???

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Any clue to this PCR question???

Postby GenePro » Oct 28 2004 8:33 pm

Dear all,

I am using 1ul of ds DNA as the template for doing a PCR for thirty cycles in a 50ul reaction volume. The concentration of the template ds DNA used is 100ng/ul. As we all know after thirty cycles there is an amplification of the template to 30 fold. How do we correlate this to the resulting concentration of the PCR product. Does it mean that 2^30 X 100ng / 50ul (reaction volume) will be concentration of the resulting PCR product?

If you have any ideas please do share..

Thanks

GP
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Postby woodshedder » Oct 28 2004 8:58 pm

No, two reasons (at least):

1) because (presumably) the region you are amplifying is only a small fraction of the template DNA. For example you may stick the whole genome in as template, but you are only amplifying a 1kb region (or whatever). So the concentration of the template DNA which will actually be copied is less than 100ng/ul.

2) also, the 2-fold amplification with each cycle is theoretical. In practice, every single template molecule will not be found by primers in every single round, especially in later rounds when the primer/template ratio is mucho decreased.

Hope that helps
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Postby Zelig_ff » Oct 30 2004 7:45 pm

Let's calculate how much DNA is 2exp30 times 100 ng...

that'd be 100 * 1073741824 ng which is about 100 grams of DNA (I suck at math, by the waym so please forgive me if I am an order of magnitude off in one direction or another). Can there be that much DNA in a 50 microliter reaction ?

:)

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not doubling every cycle

Postby Suzanne » Oct 30 2004 8:45 pm

Except the reaction is not logarithmic every cycle- which is clearly demonstrated with real-time PCR. PCR efficiency is affected by many factors including amplicon size, so as Woodshedder explained, the actual piece being amplified is much smaller and of low representation (for example a single copy gene in gDNA may be present at only a few copies in 100ng.) Also, the larger the DNA being amplified, the lower the PCR efficiency in every round, so 100% of the product from cycle 10 may not be doubled at cycle 11, and so on.

As a rough estimate, the average PCR reaction makes between 0.5-1ug of DNA for a 100 ul PCR- but I can't give you a reference for that. It's what I've been told by other knowledgable scientists. Anyone know of a reference for the ave. amount of DNA in a PCR?

Best,
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Postby Zelig_ff » Nov 01 2004 9:45 am

Under optimum conditions, PCR product will be limited by either primer or nucleotide amounts, whichever is less. It is easier to figure maximum yield based on primers - the reaction will yield as much DNA (in theory) as the molar amount of the least abundant primer.

In practice, 50 ul Pfu reaction will yield up to 2-3 micrograms, 50 ul Taq reaction may yield 5-6 micrograms provided that everything is available.

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