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How to remove DNA for quantitative PCR


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5 replies to this topic

#1 yms

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Posted 14 October 2004 - 03:38 PM

Hi,

I'm trying to see the impact of potential pcr inhibitors in my DNA extract. I'm thinking of removing the existing DNA, then spike known amount of target DNA for quantitative PCR. Does anyone know of a good way to remove the original DNA in this situation? Thanks.

#2 bob1

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Posted 20 October 2004 - 03:00 PM

Hi

You could try using a DNase and removing the DNase afterwards. The Ambion DNA-free kit used with RNA samples is pretty effective and will remove limited amounts of DNA. I suspect that the inactivation reagent in this may also remove some other proteins from solution, so it may affect the experiment you are trying.

Otherwise you could try running a blank DNA extraction, i.e. performing all the steps of your DNA extraction with no sample... there should be no DNA, but also no proteins etc carried over!

Hope this helps

#3 biomed

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Posted 22 October 2004 - 11:13 AM

Hi,

I'm trying to see the impact of potential pcr inhibitors in my DNA extract.  I'm thinking of removing the existing DNA, then spike known amount of target DNA for quantitative PCR.  Does anyone know of a good way to remove the original DNA in this situation?  Thanks.

Hi, I am kind of confused. you want to do pcr with DNA template, but why you want to remove the DNA???

#4 yms

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Posted 22 October 2004 - 03:03 PM

bob1, thanks for the response. I thought about using DNase but am not sure if I can completely remove it before spiking the target DNA for PCR. I will check with the kit and see if it works. I don't think the second method would be meaningful for what I'm trying to do though. Thanks again.

biomed, what I'm trying to do is verifying the potential PCR inhibitors in my DNA extract. What I'm thinking is to remove the extracted DNA completely, then spike with known amount of pure DNA, say E coli, then quantitatively quantify them with QPCR. Since there might have some existing E coli DNA in the sample, that's why I'm trying to remove these DNA before spiking. Hope I'm not confusing you further.

#5 Charon

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Posted 24 October 2004 - 04:05 AM

In our lab we usually remove the DNAse for subsequent qPCR reactions by simply purifying the sample again over RNA isolation columns from Qiagen.

#6 biomed

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Posted 25 October 2004 - 11:33 AM

bob1, thanks for the response.  I thought about using DNase but am not sure if I can completely remove it before spiking the target DNA for PCR.  I will check with the kit and see if it works.  I don't think the second method would be meaningful for what I'm trying to do though.  Thanks again.

biomed, what I'm trying to do is verifying the potential PCR inhibitors in my DNA extract.  What I'm thinking is to remove the extracted DNA completely, then spike with known amount of pure DNA, say E coli, then quantitatively quantify them with QPCR.  Since there might have some existing E coli DNA in the sample, that's why I'm trying to remove these DNA before spiking.  Hope I'm not confusing you further.

I got you.
I used Ambion's DNase before. It was good.
Good luck.
Biomed




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