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PCR product purification

Purification PCR products

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#1 GradMicro

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Posted 14 February 2013 - 08:16 PM

I need to PCR products that range from 200 - 600 bp. The kit used is QIAquick. i follow the default protocol with no modification. PCR volume is 50 ul, I use 5 ul to run a gel so I use 45 ul for purifcation. Bands for all PCRs are very shart/ intense. However, after purification the maximum concentration (by nano drop) that I can obtain is around 30ng/ul. Elution volume 50 ul.I need to acheive a concentration above 50ng/ul. Reducing elution volume ( i use molecular grade water) is not very practical for me as I need to obtain about 2.5 ug of DNA (thus conc needs to be > 50 ng/ul and volule 50 ul).

What I plan to do
Incubate for a longer time prior to elution
Centrifuge for a longer time.

Any other suggestion are most welcome.

Thanks a lot.

#2 GradMicro

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Posted 14 February 2013 - 08:17 PM

Forgot to mention, DNA conc mesured by Nano drop

#3 neuron

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Posted 14 February 2013 - 10:15 PM

If elution volume is critical, then more PCR reactions can be purified and pooled after purification. By doing that you will get volume more that 5oul. And there if the concentration is still less then it can be concentrated. That way your volume will still be 50ul and you can get the desired concentration also.

#4 GradMicro

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Posted 15 February 2013 - 12:02 AM

Thank you very much. However, the method that we want to try out should be a bit more sturdy in terms of having to do more samples and purify as we want to test if the method is applicable in a diagnostic laboratory. So one PCR - one puritication is the type of thing we want.
Could anyone tell me what the average purified PCR product concentration one gets from QIAquick kit when 50 ul is eluted using 50ul of ddH2O or Elution buffer? I know that this will depend on the amount of PCR product, but if the method of quantifying the PCR product is "a strong band" that is fine. (Sorry for the vague question)

#5 neuron

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Posted 15 February 2013 - 12:57 AM

OK, then In your one PCR reaction itself you should have around 80-90 ng/ul , if you want 50ng/ul after purification. And the yields of PCR products from QIAquick column are given in the manual-

http://mvz.berkeley....in_Handbook.pdf

read Agarose gel analysis of yield.

Edited by neuron, 15 February 2013 - 12:58 AM.






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