Subject: Re: Rapid genomic DNA isolation for PCR

Ma. Sheila M de Jesus msmj at edsamail.com.ph
Tue May 23 03:35:37 EST 2000


Hi Rob,

I wonder if I can request for your protocol for rapid genomic DNA
isolation for PCR from whole blood.  My next question is that can I use
frozen whole blood for this method?

This will be a big help.  I hope to get a favorable response from you.

Thank you!

Sheila
(boston at quickweb.com.ph)
Philippines
-----------------------
owner-methods at hgmp.mrc.ac.uk wrote:
>Eric Rheaume wrote:

>> I am looking for a very rapid genomic DNA obtention method suitable for PCR
>> analysis. That method must be fast (for up to 200 samples), efficient (we
>> want to detect one clone in 100 from 100000 cells) and must work with cell
>> lines. We have been using a PCR buffer with nonionic detergents (KCl, Tris,
>> NP40, Tween20) and proteinase K method with limited success due to much
>> variability. Anyone out there using routine procedure that would be more
>> reproducible?

>I'm using a pretty good method that was originally written for DNA isolation
>from ~9 ml of whole blood.  This was very large scale and the obvious
>bottleneck in the procedure was the centrifugation steps.  I could only do 4
>samples at a time (50 ml conical tubes).  I scaled it down to 1 ml blood and
>centrifugation in 15 ml conical tubes and there is no reason to suspect that
>this couldn't be scaled down to 100 microlitres of blood and centrifuged in 1.5
>ml Eppendorf tubes.  From the 1 ml of blood, I resuspended the DNA in 300
>microlitres TE and used 1 microlitre in PCR.  This DNA is quite concentrated
>and so I could likely get away with resuspending it in a larger volume.  My
>point here is that even from 100 microlitres of blood, you could probably get
>30-50 microlitres of DNA for PCR.  The DNA is very good quality for my
>purposes, and does not use any fancy chemicals, columns or equipment.  From
>start to finish, the procedure takes about 5 hours (I think, I've never timed
>it) and after lysing the blood cells, the samples are safe at 4 degrees for a
>couple of weeks, so the procedure can be easily split up if need be.  If you
>want the protocol, let me know.  I can't say for sure that it would work at the
>level of 100 microlitres of blood, but I also don't see why it wouldn't.  Again
>the limitation would be the number of slots in your centrifuge.
>
>This may not be the answer you're looking for, but I thought I'd throw it out
>there in case it helps.
>
>Rob
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