I have clotted, frozen avian red blood cells that I am attempting to extract DNA from (for eventual micro satellite sequencing). I am using the Qiagen DNeasy Blood and Tissue Kit. The protocol was not working (low DNA yield and poor quality), so I have modified the lysis step: frozen blood (0.02+g), add 250mL ice cold 90% ethanol, the lysis buffer and protinease k and mix it all using a tissue homogenizer; then incubate it at 56C for over an hour, vortexing the samples every ~20min and again using the homogenizer about half way through, but my cells are still not lysing.
Does anyone have any suggestions?
Thanks in advance!
Hi,
How about this: http://www.tataa.com/wp-content/uploads/2012/10/prepGEM_Blood_and_StorageCard_AppNote_AvianBlood.pdf ?
What do you call tissue homogeneiser ? A Dounce ? Did you check the grinding diameter of your device ? You could alternatively look for a bead beater
Hi,
I would recommend incubating for at least 12 hours during the lysis step, if not over night. Given that you're targeting microsatellites you could probably get away with using a cheaper extraction protocol such as salting out - I find it yields more DNA than kit-based methods anyway. You can find a protocol here: Article Population diversity and relatedness in Sugarbirds (Promerop...
All the best
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts