Problems in the DNA extraction from blood of rats - (Apr/09/2007 )
Please i want to solve my problem concerning the extraction of DNA from blood of rats
at first in my research i take a blood sample from rat by retro-orbital bleeding then i need the same rat to be alive as i will give it a toxic dose then take another blood sample
the first problem:
is that we anaesthetize the rat with chloroform - and the rat die immediately
is there any method to anaesthetize the rat and keep it alive?????
the second problem
when we collect blood from eye into a tube containing around 100ul of EDTA(0.5M) the blood is clotting
i dont konw the reason for that
the third problem
when we do DNA extraction from the blood by salting out method - when we do washing by red blood cells lysis buffer the pellet remain red after several washing so i cann't complete the remaining steps
PLEASE help me to solve my problems
Best Regards
Hi, I hope I can answer some of your questions.
is that we anaesthetize the rat with chloroform - and the rat die immediately
is there any method to anaesthetize the rat and keep it alive?????
Try using Ether or an ip anesthetic like thiopental or pentobarbital.
when we collect blood from eye into a tube containing around 100ul of EDTA(0.5M) the blood is clotting
i dont konw the reason for that
Do you use capillaries for the collection? Then you need to have them filled with EDTA or Citrat, also., If you use them like that you start the clotting cascade before the blood is in cotact with the EDTA.
when we do DNA extraction from the blood by salting out method - when we do washing by red blood cells lysis buffer the pellet remain red after several washing so i cann't complete the remaining steps
I have allways used a column extraction (DNeasy blood and tissue kit) -mever had any trouble!
Krümel
Thank you very much for your thoughtful reply
please i would greatly appreciate clearifying some points for me
I use capillaries filled with EDTA in the collection. but the clotting occur
Also please explain to me what is (DNeasy blood and tissue kit)?????
and tell me the name of the company which sell it
Thanks very much. Best Regards.
Clotting - strange, it normally works for me?
DNeasy blood and tissue kit is a column based DNA extration kit from QIAGEN.
K.
Thanks very much.
K.
PLEASE tell me what the concentration and the amount of EDTA you use in the collection.
Best Regards.
PLEASE tell me what the concentration and the amount of EDTA (and reciep) which used in the collection of Blood from retro-orbital site.
Best Regards.