1. Draw 5 ml of blood using lavender-top Vacutainer (Beckton-Dickinson; EDTA anticoagulant)
2. Keep cool until prep is performed (e.g. in an ice chest), but DO NOT FREEZE. Highest yield will be achieved by extracting within 24 hours.
3. Empty blood into a 15 ml Falcon tube; add 10 ml of Red Cell Lysis Buffer (RCLB) and mix completely by inversion.
RCLB: 1 mM NH4HCO3
115 mM NH4Cl
Note: You can bring pre-weighed NH4Cl and a 1M solution of NH4HCO3, and use the cleanest water available to make RCLB on-site. Try to use distilled water, but in a pinch I suspect that water from a personal filter (e.g. Pur, etc.) would work. If it is to be used immediately, this solution does not need to be autoclaved; if you plan to store it for more than a day, autoclave.
4. Spin 10 minutes @ 1,200g in a clinical centrifuge.
5. Discard supernatant and resuspend cell pellet in 10 ml RCLB; repeat step 4.
6. Discard supernatant; resuspend cell pellet (it should be white now) in 1.8 ml of White Cell Lysis Buffer (WCLB). It should be extremely gelatinous – like snot.
WCLB: 100 mM Tris-Cl (pH 7.6)
40 mM EDTA (pH 8.0)
50 mM NaCl
0.05% Sodium azide
Note: This solution (before the addition of SDS) definitely needs to be autoclaved – this means bringing a heavy, well-protected bottle with you, or being completely certain that you will be able to autoclave on site. Add SDS after the autoclaved solution cools. Remember that the DNA is going to sit around in this stuff under less-than-ideal conditions for a considerable period of time…
7. Store each white cell lysate in a 2 ml screw-top tube. DNA should be stable in this form for several weeks at room temperature, although refrigeration is recommended just to be safe.
8. To perform final extraction, add 150 µl of saturated NaCl (~ 6M NaCl) to 400 µl of white cell lysate.
9. Vortex and invert to mix; place on ice for 10′.
12. Centrifuge 5′ @ 12,000 RPM.
12. Add the supernatant to a 1.5 ml tube (550 µl); add 1000 µl absolute (100%) ethanol. Mix by inversion – the DNA precipitate should be visible at this point.
13. Spin 5′ @ 12,000 RPM; discard supernatant.
14. Wash w/ 1 ml 70% ethanol; air dry and resuspend in 100 µl of dilute TE (1:4 w/ H2O). Leave overnight at 4°C to resuspend completely. Check concentration and dilute to 100 ng/µl for stock. Freeze white cell lysates at -70°C for long-term storage.
Recommended field equipment for DNA extraction
1. Clinical centrifuge capable of at least 1,000 RPM, or hand-cranked model
2. 10 ml pipettes (minimum of 1 per extraction session – e.g. 1/day)
3. 15 ml Falcon tubes (1 per sample)
4. P-1000 Pipetteman
5. 1000 µl pipette tips (minimum of 1/sample)
6. 2 ml screw-top tubes
8. Tube racks (for Eppendorfs and 15 ml tubes)
9. Blood collecting supplies (Vacutainers, needles [21 gauge if possible], plastic housings, tourniquets, alcohol preps, cotton, Band-Aids, biohazard bin for needles)
10. Ice chest (pack the other supplies inside for travel – and bring documentation for customs in case you are asked about all of those needles, white powders and solutions inside)
https://protocolpedia.com/wp-content/uploads/2017/01/LOGO-pp.png00Editorhttps://protocolpedia.com/wp-content/uploads/2017/01/LOGO-pp.pngEditor2017-05-11 14:15:452017-07-03 12:52:25Dna Extraction Procedure From Human Blood
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