2 replies to this topic

[mightymus]

I need your suggestion for isolating DNA from the blood sample which we want to use in our study. This is what I was planning to do for extraction.

Centrifuge the blood sample and extract the cells and then homoginise the cells using bullet blender. After that I follow the QUIGEN protocol for extraction of DNA. Has anyone done something like this? If so, can you tell me how effective it can be. If this is not a good method, can you suggest a simple and effective way? We need the DNA for our methylation studies. Thanks in advance (:

--mightymus--

[Trof]

First get rid of erythrocytes. There is huge amount of them and heme is a potent PCR inhibitor. We lyse the erythrocytes from whole EDTA blood (heparin is also not suitable for PCR later) and use only washed leukocytes for DNA isolation.

5-9 ml of blood fil up to 50 ml with 1x lysis buffer (ice cold) and mix.
40 minutes on ice, mix gently once in a while.
centrifuge 10 minutes/ 1125 g/ 4 deg
draw off supernatant, wash (resuspend pelet) with 15 ml of 1x lysis buffer
centrifuge again
wash with 15 ml ice cold PBS pH 7.4
centrifuge again
now you should have (mostly) leukocytes, for any subsequent DNA isolation method.

10x lysis buffer
1.5 M  NH₄Cl   
100 mM NH₄HCO₃   
10 mM Na₂EDTA
adjust pH to 8.

But if you use QIAGEN kits you may as well buy QIAGEN kit designed for blood, then you don't need to do this.

[mightymus]

Thanks Trof. I tried your method but unfortunately I could get only 1.2ng/µl . I should have mentioned before itself. The blood sample we have is frozen. So we are planning to try the Ficoll method now. We are planning to prepare our own Ficoll plaque solution. Can I ask if it is possible to skip Sodium Diatrizoate since we dont have it. How much will that effect our extraction if in case we sont use it?