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DNA from old frozen and partly coagulated heparin blood

DNA from old frozen and partly coagulated heparin blood - Protocols and Methods Forum

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  #1  
Old 03-29-2006, 10:42 PM
Deanne Bell
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Default DNA from old frozen and partly coagulated heparin blood



Dear Andy

This is just a thought, do you really need to do DNA extraction?? Can
you try your probe / PCR using the frozen blood?

I don't have much experience with blood and don't know exactly what is
meant by "200 probes, with clinical data available", but I have done a
PCR straight off a frozen suspension of bacteria cells with great
results.

HTH
DBell

-----Original Message-----
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[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of AndyFrey
Sent: Wednesday, March 29, 2006 12:20 PM
To: [Only registered users see links. ]
Subject: DNA from old frozen and partly coagulated heparin blood

Hi,
my standard method for extraction does not yield any DNA from this
frozen heparin blood. I also tried the Quiagen QuiAmp Midi Kit but with
no result.

Counting the WBC yielded approx. 600/ul, probably due to freezing?
Qualitative observation with Giemsa coloring, showd no intact nuclei of
WBC beside the counted cells.

Probably the problem could be that the probes are partly coagulated due
to insufficient mixing of the li-heparin blood container?

Any suggestions are welcome, the problem pertains around 200 probes,
with clinical data availiable. So it's very important for me to find a
way to extract the DNA.

Yours, Andy

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  #2  
Old 03-30-2006, 03:28 AM
AndyFrey
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Default DNA from old frozen and partly coagulated heparin blood

Hi, I meant:
200 "samples" with appropriate data about phenotypes are a considerable
treasure for us, which we won't loose by discarding them.

Normally we archive them as isolated DNA at -20C, also for exchange
with other labs the standard "isolated DNA" would be necessary.

But in case of no return, Your idea is an option of course,
Thanks, Andy

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  #3  
Old 03-30-2006, 07:28 PM
marc crepeau
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Default DNA from old frozen and partly coagulated heparin blood

I'd be curious to know how you determined that there was no DNA.
Agarose gel? Spectrophotometry? Also, are you using PCR based methods
for your genetic analysis? In my experience, DNA from
heparin-preserved whole blood is useless for PCR since the heparin
inhibits the reaction and is impossible to remove. But Novagen sells a
product called BloodDirect that they claim allows PCR directly from
whole blood stored fresh or frozen with a variety of anti-coagulants,
including heparin. I've never tried it, but you might want to look
into it. Good luck!

-Marc

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  #4  
Old 04-01-2006, 05:16 PM
AndyFrey
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Default DNA from old frozen and partly coagulated heparin blood

marc crepeau wrote:

My statement referred to the method we're using normally. This ist a
standard method of selective lysis of RBC then lysis of the nucleoli,
Protein extraction and DNA precipitation in EtOH. This means that, its
important to have enough solved DNA before precipitate it, if not there
will no precipitation be seen, by eye. This in conclusion means that
the method failed to yield DNA in this case.

We now, switched the method and instead apply the Quiagen QuiAMP DNA
Blood Kit. It works even in this case, it also promises to work with
heparin Blood well. Because it consists of an ionic-exchange column
where the DNA is bound until the last eluation step i *hope* that the
DNA product will contain no heparin or maximum a tolerable amount of
it.

Yours Andy

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