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DNA isolation from whole blood + clotting??!!


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#1 Haley

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Posted 18 November 2013 - 06:13 AM

Hello!

 

For demonstration purposes, I sometimes need to prepare DNA from whole blood through a very "rough" procedure. The blood is collected with a butterfly needle in 10 ml-heparin tubes, and then I transfer this 10 ml of blood into a 50 ml-falcon tube. To this, I add 40 ml of red cell lysis buffer, I incubate on ice for 10 minutes, and then I centrifuge on 3000 rpm for 10 minutes. Afterwards, you should see a nice white pellet at the bottom of the falcon tube. This is where it goes wrong too often to be a freak accident. In about 50% of the cases, I don't get a nice pellet, but a thick red clot is formed in the bottom of the falcon tube, with red liquid above it. It is like the heparin didn't work and the blood still clotted. But how can that be, because there was heparin in the tubes, and from the same batch of collected blood, there will be some falcon tubes that do have a nice pellet. It's like it's a completely random event, but very annoying when you need to demonstate something. Can anybody shed some light on this mystery??



#2 bob1

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Posted 18 November 2013 - 11:44 AM

Make sure you mix the tubes adequately when collecting the blood - inverting 10-20 times should work.



#3 Haley

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Posted 19 November 2013 - 12:40 AM

Thank you!

 

I also heard another possibility. Someone told me it could be due to the fact that, using butterfly needles, the blood will first need to go through some narrow tubing before it enters the heparin tube. This could cause the blood to start clotting already, because it doesn't immediately enter the heparin tube. Do you think this could be a reason?






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