Manual DNA Extraction Method from Human Blood

1. Collect whole blood in a Vacutainer tube (purple-stoppered) containing 100 μl of 15% EDTA.
2. 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 MgCl_2, and 2 mM EDTA (TKM1). [Note: Mix the contents of the tube thoroughly]
3. 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 the solution].
4. Centrifuge at 2200 RPM for 10 min at room temperature (RT) in a Beckman table-top centrifuge.
5. 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]
6. 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).
7. Add 50 μl of 10% SDS, mix the whole suspension thoroughly by pipetting back and forth several times, and incubate for 10 min at 55°C.
8. 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].
9. Centrifuge at 12000 RPM for 5 min, in microcentrifuge.
10. Save the supernatant containing DNA and discard the precipitated protein pellet at the bottom of the tube.
11. To the supernatant, add an equal volume of isopropanol at RT and invert the tube several times until the DNA precipitates.
12. Centrifuge for 5 min at 12000 RPM at 4°C.
13. Discard the supernatant, leave the pellet for drying, and re-suspend DNA in 0.5 ml of Tris_EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Perform Agarose Gel Electrophoresis.

Note: The above procedure can also be done with a smaller volume of blood with a proportional decrease in the buffers and other chemicals.

Reference:
Lahiri, D. K., & Nurnberger Jr, J. I. (1991). A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic acids research, 19(19), 5444.

Share

Related posts:

Plasmid DNA Extraction from Bacteria by Alkaline Lysis Method How to reduce the number of screenings for transformed bacterial colonies? Manual Technique for Extraction of DNA from Bacteria Underlying Principle of Different Solutions Used for Isolation of Plasmid from Bacteria by Alkaline Lysis Method Factors affecting migration of Nucleic Acids in Agarose Gel Electrophoresis