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DNA isolation/purification troubleshooting.


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5 replies to this topic

#1 Wek

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Posted 20 May 2012 - 01:14 PM

I'm having some trouble isolating and purifying DNA from tissue. At first I was getting a bad A260/A280 (~1.2-1.5) because I was taking DNA from the upper layer of the aqueous phase instead of the middle. But now I'm getting a high A260/A280(~2.0-2.2). It's not the protocol I'm using since it's been optimized for our purposes. I have been able to get a ~1.8-1.9 A260/A280 once (out of three tries) but that was after a phenol chloroform cleanup. That's time consuming and tedious so I'm looking for tips on what I could be doing wrong without knowing it. My last try at it I had a problem getting an accurate A260/A280 from the spectrophotometer, which has been used for this work for a long time. Needless to say, my PCR didn't work except for the controls.

Btw, what happens when you use a slightly high (~75%) or slightly low (65%) EtOH for washing?

#2 bob1

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Posted 20 May 2012 - 01:56 PM

Which of the many DNA isolation protocols/reagents are you using?

#3 Wek

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Posted 20 May 2012 - 02:01 PM

The protocol uses TNES and TE to resuspend DNA.

Similar to this protocol.
http://www.genomics....CH/ISOLATIO.PDF

Edited by Wek, 20 May 2012 - 02:02 PM.


#4 bob1

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Posted 21 May 2012 - 12:55 PM

OK, if it is that protocol, you can always expect some protein to be coming over with the DNA, especially the proteinase K. If you need pure DNA, add at least two phenol-chloroform extractions after the digestion steps. Yes, they are time consuming, but if you need clean DNA...

You can also do column extractions (from a kit) of DNA that will give you clean DNA, but they are relatively expensive.

Also, if you have too much EDTA in your samples, this will inhibit the PCR by chelating the Mg ions needed as co-factors for the DNA polymerase. Try diluting your samples 1:100 or 1:1000 to see if that works.

#5 Wek

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Posted 21 May 2012 - 05:24 PM

I have done column extractions but the amount of DNA samples we have to isolate and purify is too much to so column extraction will become very expensive.

I will try diluting the samples first and see if that works. If not then I'll do a couple of phenol extractions.

Also, will using a lower percentage of ethanol (65%) for the wash steps affect the DNA purification process?

#6 bob1

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Posted 23 May 2012 - 12:51 PM

Note that I meant that you should dilute the extracted DNA rather than the sample prior to extraction, which would just make it harder to extract the DNA.

The percentage ethanol is designed to keep the DNA precipitated but still dissolve salts. I don't think 65% instead of 70% would have too much effect, but I can't be sure on that.




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