DNA extraction from avian blood - (Feb/14/2001 )
Here is a protocol for extracting DNA from blood without using a kit, but I am not sure it's suitable for avian blood.
a. Collect the blood sample into EDTA tube and freeze at –20C.
b. Thaw the sample and transfer a 200ul aliquot to 800ul of freshly prepared 170mM NH4Cl in a microcentrifuge tube. Rotate the tube for 20min to mix the contents thoroughly.
c. Centrifuge the sample for 2min in a microcentrifuge to obtain a whit cell pellet and discard the supernatant.
d. Wash the cell pellet with 300ul of 10mM NaCl, 10mM EDTA pH7.5, and collect the pellet by centrifugation for 15sec. Repeat the washing step three more times to remove all visible haem.
e. Resuspend the final cell pellet in 500ul of 50mM NaOH by vortexing for 10sec.
f. Incubate the sample at 100C for 5min.
g. Neutralize the sample with 100ul of 1M Tris-HCl pH7.5 and vortex the mixture for 5sec.
h. Centrifuge the sample for 15sec to remove cell debris and retain the supernatnat containing the DNA.
i. Store the DNA solution at –20C until use.
Reagents EDTA tube 170mM NH4Cl (freshly prepared) 10mM NaCl, 10mM EDTA (pH7.5) 50mM NaOH 1M Tris-HCl pH 7.5
What is the best protocol for the extraction of DNA from avian blood without ordering a kit. If possible please include exact protocol.Thank you,Erica