how to extract DNA from frozen blood ? - for help (Mar/11/2008 )

hello,everyone !
I want to extrat genomic DNA from Frozen Blood. The blood has been stored about two years at subzero 80 degree. What should I do ?
Any suggestion or protocol is invited ! Thank you very much !

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We extract genomic DNA from whole blood using the wizard DNA extraction kit from promega. We've only used fresh blood but i'm assuming it could work for frozen as well. The kit allows you to use anywhere from 300ul to 3ml. Good luck!

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We used protocol from this site
http://www.protocol-online.org/prot/Protoc...blood-3171.html
However, you need to make sure you save your blood in correct way (I believe no heprin).

No phenol Chloroform needed in this protocol, and it works fine, around 15 ug DNA from 1 ml of frozed blood when we did it.
You should at least take look it.

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In addition, Because it has been stored about two years, Hemolysis was occurred in all of the blood. Now I can't find any blood cell in microscope.
In this situation, whether can I extract genomic DNA from these blood ? how ?

thank you !

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My samples also froze in -20 for a years (couple of them from 1988), and all samples are still produce DNA.

My understanding is DNA extraction requires white blood cells and Hemolysis occured on red blood cells, which do not contain DNA (OK do not contain genomic DNA). The only thing is to wash pellet hard before proteinase K step.

Good luck

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Thank you very much ! I'll have a try.

Any suggestion is also invited now.

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Hello!
I have the same problem...I must to exstract Genomic DNA from frozen (-18 for 4 years) that I thaw out 1 year ago to perform an extraction whit comercial column kit (Sigma...8 ug of DNA from 200ul of whole blood).
Now I ned to obtain a total of >10 ug of DNA fromthe same samples....I try this method and I have good results..

1-thaw out the bloods samples incubating at 20° C;
2-Strore the sample in ice;
3-pipette 500 ul of whole blood in a 2ml tube;
4-add 500 ul of lysis buffer (tris, EDTA, SDS ph8) + 10 ul Proteinase K(20mg/ul);
5- seal up whit parafilm (important!!) and incubate at 55°/60° C for 2 hours;
6- boil the samples for 10 minutes;
7- centrifuge at 12000 xg speed for 3 min;
8- add 1 vol of phenol:chloroform:isoamyl alcohol (I am trying now with simple ammonium acetate protein, appen Gentra® Puregene® Handbook ...work in progres )precipitation;
9-centrifuge 12,000g 10min
10- transfer upper aqueous layer to new tube, add 0.1Vol 3M NaAc and 2.5Vol 95% Etoh to precipitate;
11- 12,000g 3 min
12- wash the pellet (may be not visible) with Etoh 70%;
13- elute in 100-200 ul TE
...let me know if you have results
Regards.

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