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Genomic DNA extraction - how to quantify for PCR?


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9 replies to this topic

#1 biologg

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Posted 29 October 2012 - 11:32 AM

Hello,

I would like to ask you the following: I need to extract genomic DNA from 2 different cell types, run a PCR to amplify an approximately 900bp fragment and send the PCR amplicon for sequencing. Can someone please tell me, as I am quite inexperienced in this:

After genomic DNA extraction, what would be the best way to quantify the amount/concentration of DNA I have, in order to load it on the PCR plate? Can I use nanodrop, for example? Someone told me nanodrop is not a good idea, as following extraction, my samples will contain lots of RNA and i will not get correct measurement. But this maybe depends on the protocol for genomic DNA extraction? Can you recommend me a protocol?

Overall, this is what i would like to do:

1) extract genomic DNA from cells
2) determine DNA concentration
3) run PCR to amplify sequence
4) run the product on a gel and purify it
5) send for sequencing

I would appreciate it if you can give me some advice or if you think i should do some additional steps.

Thank you!

#2 bob1

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Posted 29 October 2012 - 11:46 AM

There's no way to easily distinguish between genomic DNA and RNA on a spec (e.g. nanodrop) - but there is another method that you should know about (hint - it's run in tanks) that will give you some idea of the level of RNA contamination and whether you can trust the concentration given by a spec. I don't know of any easy method that will reliably give a concentration of DNA in a solution that is contaminated with RNA.

A good genomic DNA isolation protocol shouldn't yield much RNA, especially with a bit of practise.

#3 biologg

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Posted 29 October 2012 - 11:59 AM

Thank you, bob1! So basically, i can still go on and measure the concentration by nanodrop and run the PCR despite maybe having a bit of RNA in the sample? I was thinking to use this extraction protocol:

http://www.protocol-...issue-1157.html

#4 bob1

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Posted 29 October 2012 - 12:07 PM

That protocol will work, but is quite likely to give you high levels of RNA. If you want to minimize RNA, use a phenol/chloroform extraction of the appropriate pH (pH is primarily what determines if you get RNA or DNA). You will need to run the samples on a gel to see if there is a lot of RNA in them.

#5 hobglobin

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Posted 29 October 2012 - 12:11 PM

another way would be to add RNase during the DNA extraction or later to the buffer ("RNAse in TE") with the DNA and then do another clean up by ethanol precipitation or with a column...

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#6 biologg

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Posted 29 October 2012 - 12:36 PM

I was also thinking of RNAse treatment, so i think i will probably go with it. However, the presence of Proteinase K in the buffer would also degrade the RNAse, wouldn't it? So in this case, should i add it in the TE buffer in the end? Do you know if i need to incubate with RNAse for specific period of time to make sure i degrade all RNA, or does it act in the matter of seconds?

#7 hobglobin

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Posted 29 October 2012 - 12:44 PM

We usually add it in the buffer after the extraction with a very low concentration (and is does not disturb our downstream applications, usually PCR).
Or you add it to the lysis buffer but before the Proteinase k and give it some time, RNase is quite stable and should stand e.g. SDS for some time if it's in the buffer...

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#8 Trof

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Posted 29 October 2012 - 12:46 PM

Most probably PCR would just work, if you add about 100ng of what you have. High concentration of RNA may inhibit PCR, but probably those who made that protocol needed to work with the DNA too, and PCR is very likely to be involved. So I would just try the PCR before anything else (if you know you have a working PCR protocol or some other better quality DNA to optimise it first).

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#9 hobglobin

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Posted 29 October 2012 - 01:03 PM

Most probably PCR would just work, if you add about 100ng of what you have. High concentration of RNA may inhibit PCR, but probably those who made that protocol needed to work with the DNA too, and PCR is very likely to be involved. So I would just try the PCR before anything else (if you know you have a working PCR protocol or some other better quality DNA to optimise it first).

I heard this too once but still have no idea about the mechanism and from which concentration RNA might inhibit...has anybody an idea?
When I tried out my PCRs with RNase treatment and without there were never a difference visible (on a gel).

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#10 biologg

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Posted 29 October 2012 - 01:13 PM

Considering your comments, I guess the best thing to do in my case would be to add RNAse to both lysis buffer in the beginning and to the TE buffer in the end, then measure with nanodrop. In this case, hopefully, i will not have high amount of RNA to interfere with the PCR reaction (whatever the reason for that may be) and will be able to amplify my DNA sequence. Then, i can just run the PCR product on a gel and purify it for sequencing. I guess in this way i should get minimum RNA contamination (with a bit of practice maybe). By the way, they told me to be careful when purifying the amplicon from the gel, as silica or resin might interfere with sequencing as well. Have you heard anything like that?

Edited by biologg, 29 October 2012 - 01:14 PM.





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