Dear all,
My lab was isolated leukocytes and stored at -20C. And now I am trying to isolate DNA from it by using Zymo DNA isolation minipreb kit.
I encountered several problems, first I could not resuspend the pellet of cells. Eventhough after lysis and proteinase K threatment, there is some pellet remained. we thought that maybe it was because of coagulation and we used lysis buffer for it. And I tried tissue lysis buffer in the kit as well. And also I increased the time of incubation at 55C. But these solutions did not work for me.
I could not isolate the DNA. Maybe I thought the paticules or pellet part could block the matrix in the column. Or 1 year could affect the DNA eventhough I am not positive about it.
So What do you think about my problem? How can I isolate DNA ?
Same blood sample PBMC's are also isolated but I did not have any problem isolating DNA from PBMC by using the same kit.
EDIT: I've tried overnight or 24H Proteinase K and lysis buffer digestion and I increased the vortex step like 1 minute. After that I could obtain enough DNA.
Thanks in advance.
Suleyman
was PBMC freeze in culture medium plus DMSO? If PBMC were frozen in some medium/buffer i recommend you that once you thaw your cells, centrifuge and resuspend in a minimum volume of PBS before adding lysis buffer
Dear Suleyman Bozkurt,
In our lab we have experience of DNA extraction from old blood samples using the routine phenol-chloroform method, and it was work perfectly. You can try this approach in your experiment.
Best regards,
Maxim
Dear Eddy and Maxim,
Thank you for your sincere response. I've tried overnight or 24H Proteinase K and lysis buffer digestion and I increased the vortex step like 1 minute. After that I could obtain enough DNA. after all we can obtain enough DNA after 1 year. Maybe this information may help other people later on.
Thanks
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