Calculating DNA concentration in PCR product

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claudiupopa
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Calculating DNA concentration in PCR product

Post by claudiupopa » Sat May 02, 2009 3:18 pm

I need to figure the concentration of amplified DNA after a PCR by using available information like concentration of template DNA, enzyme speed, number of PCR cycles and so forth. Someone told me that i should be able to figure it out myself but im really bad with numbers and math so i got nowhere. Is there any software or some math equation or procedure that i can follow to the above mentioned effect?
Thanks a bunch

shahensha
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Post by shahensha » Sun May 03, 2009 6:26 am

simply by using uv spectrophotometer you can estimate the concentration of dna after pcr amplification

claudiupopa
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Re: Calculating DNA concentration in PCR product

Post by claudiupopa » Sun May 03, 2009 9:42 am

yes but the spec can be set off by other components in the pcr mix, like leftover primers and nucleotides. Any way to circumvent that? i need accurate readings of the concentration

smellor
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Post by smellor » Wed May 13, 2009 8:12 pm

well a good rule of thumb is 2^n where n is number of cycles (provided you have a single dsDNA molecule to start with). so after 10 cycles e.g. 1024 molecules, but i don't know if that is sufficient for you...

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mith
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Post by mith » Fri May 15, 2009 9:03 pm

You'd just use the doubling formula as smellor described(tweak it for your template concentration). Ideally it doubles, of course there's diffusion and other effects
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DanielSan
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Post by DanielSan » Sun May 17, 2009 8:26 pm

You could use a fluorometric dye such as Hoechst33258. It is rel. cheap (around 20$/100mg) and it specifically binds to dsDNA. It has a linear range from at least 50-800 ng DNA/mL. With special assays you can go down to 5 ng/mL.
Other options would be Ethidium bromide (which is around 10 times less sensitive) or the fluorescent dye SybrGreen.

jiajia1987
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Post by jiajia1987 » Thu Jun 04, 2009 8:51 am

Alternatively, use a Nanodrop to find out the concentration of the PCR products!

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