I am writting down the protocol for the DNA Isolation via Salting out process from blood. Gives good yield of DNA. A260/280= 1.6-1.8 (best)
Reagents
1) Solution A Haemolysis Solution * Prepare fresh or use a solution prepared in 24 hrs and stored at 4'C.
Final Volume (ml): 1000 750 500 100
Sucrose (g): 109.5 82.125 54.75 10.95
1M MgCl2 (ml): 5.0 3.75 2.5 0.5
Dissolve the above in autoclaved water. Do not autoclave after dissolution as it can char the sucrose.
Add Triton X @ 1% of the final volume.
2) Solution B WBC Lysis buffer * Prepare fresh or use a solution prepared in 24 hrs and stored at 4'C. Add SDS prior use.
Final Volume (ml): 100 50
1M Tris-HCl;pH ~8 (ml): 40 20
0.5M EDTA (ml): 12 6
1M NaCl (ml): 15 7.5
Dissolve in dd.water. Autoclave and then add 20% SDS @ 5% to the final volume.
3) Solution C Protein ppt. buffer * Light sensitive, so store in amber bottle at 4'C; storage can be done for 1 year at the max.
This solution is 5M Sodium perchlorate= 70.23g of sodium perchlorate in 100 dd. water/ autoclaved water.
4) 0.9% NaCl (mammalian saline)
5) for 200ml TE: 2ml 1M Tris/Trizma(Sigma) + 0.4ml 0.5M EDTA. pH: 8.4
6) Cloroform (in 4'C)
7) 70% Ethanol (in 4'C)
8) Isopropanol (in 4'C)
Procedure
S1. To 1 volume of heparin added blood add 5 volumes of 0.9% NaCl. Mix by inversion. Centrifuge at 2000rpm for 10 minutes.
S2. Decant the supernatant.
S3. Add solution A- 4 vols of the original blood taken and mix slowly by inversion. The solution will turn clear red due to lysis of RBC. Spin at 3500rpm for 5 minutes. If a white pellet is clean, proceed to next step or else any red pellet indicates incomplete RBC lysis. Add 2ml of Solution A and repeat the S3. Discard the supernatant.
S4. Add 2ml of Solution B, vortex to dislodge pellet. Invert mix for 10minutes unless the pellet looses it's integrity. (To hasten the process, I suggest to keep the mix in 37'C for 5 minutes in between two inversions of 7 minutes and 3 minutes.) If the lysis of WBC takes place, the pellet looks slightly thready and the solution gains a silky appearance of khadi silk.
S5. Add 500uL of solution C and mix rigorously using vortex.
S6. Add 2ml of chilled chloroform and mix. The solution turns milky.
S7. Centrifuge at 2500rpm for 5 minutes.
S8. Transfer the clear aqous layer in 1.5mL tubes. This may take several turns to transfer all the Aq. supernatant. The suggested process:
S8/1: Take 500uL of supernatant and add 500uL of Chilled isopropanol, Invert mix by hand. Appearance of translucent threads. These are alcohol insoluble DNA.
S8/2: Spin down at 13000rpm for 5 minutes or 10000rpm for 12 minutes.
S8/3; Keep the pellet (DNA) and discard the supernatant. Repeat S8/1 unless all the aqous layer from S7 spin is accumulated in one 1.5mL tube for a particular sample.
S9. Add 1ml of chilled 70% ethanol. Wait for 5 minutes and spin at the speed conditions of S8/2.
Discard the alcohol and air dry. (a maximun dry of 18 hours is allowed; overnight dry). Keep in mind, not to over dry!
Add 100uL TE to the air dried pellet. Keep in 4'C for 3 days, before estimation and analysis.
Hope this will help a lot of my molecular biologists in different arena of life science. This is a tried and tested protocol.
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