Protocol Series: Extraction of DNA from Blood [Modified Lahiri and Nurenburger Method]
The protocol listed below is a modified version of the method devised by Lahiri and Nurenburger in 1991 for extraction of DNA from blood. The original article can be summoned from the web. The methodology described here has been optimized for the laboratory of Central Department of Biotechnology, Tribhuvan University. The protocol can be utilized for blood amount as little as 500 microliters with proportional decrease of the components.
- Collect whole blood in a Vacutainer tube (purple-stoppered) containing 100 μl of 15% EDTA.
- Transfer 5 ml of blood into a 15 ml centrifuge tube and add 5 ml of low salt buffer containing 10 mM Tris-HCl pH 7.6, 10 mM KCl, 10 mM MgCl2 and 2 mM EDTA (TKMl). [Note: Mix the contents of tube thoroughly]
- Add 125 μl of Nonidet P-40 (NP-40) to lyse the cells. Mix well by inversion several times. [Note: Mix the solution until NP – 40 gets dissolved in he solution].
- Centrifuge at 2200 RPM for 10 min at room temperature (RT) in a Beckman table-top centrifuge.
- Slowly pour off the supernatant and save the nuclear pellet (the small pellet at the very bottom of the tube) and wash the pellet in 5 ml of TKM1 buffer and centrifuge as before.[Note: If the pellet remains red, wash with TKM1 repeatedly]
- Gently resuspend the pellet in 0.8 ml of high salt buffer containing 10 mM Tris-HC1 pH 7.6, 10 mM KCl, 10 mM MgCl2, 0.4 M NaCl and 2 mM EDTA (TKM2).
- Add 50 μl of 10% SDS and then mix the whole suspension thoroughly by pipetting back and forth several times and incubate for 10 min at 55°C.
- Add 0.30 ml of 6 M NaCl in the tube and mix well. [Note: Do not Vortex as it can cause Shearing of DNA].
- Centrifuge at 12000 RPM for 5 min, in microcentrifuge.
- Save the supernatant containing DNA and discard the precipitated protein pellet at the bottom of the tube.
- To the supernatant add 2 volumes of 100% ethanol at RT and invert the tube several times until the DNA precipitates.
- Remove the precipitated DNA strands and put them in a microcentrifuge tube containing 1 ml of ice-cold 70% ethanol.
- Centrifuge for 5 min at 12000 RPM at 4oC
- Leave the pellet for drying and re-suspend DNA in 0.5 ml of 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
- Perform Agarose Gel Electrophoresis with 0.8 % Agar.
Note: When decreasing the blood volume to 500 μl , use 15 μl of NP – 40 , 100 μl of TKM2, 10 μl of SDS in their respective steps. Similarly, 2 volumes of 100 % ethanol can be replaced with equal volume of isopropanol. Although recommended to be carried out at 4oC, the entire experiment can be carried out at room temperature as well. The genomic DNA thus can and has been successfully utilized for Polymerase Chain Reaction (PCR).
Note: Please leave a comment if you have any queries regarding the protocol and we shall try to provide you with answer at the earliest.
Lahiri DK and Nurenburger JI (1991) A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Research. 19 (19): 5444.