Genomic DNA Isolation

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"Protocol1. Take 10ml of blood sample.2. Add 35ml of Tris-EDTA buffer to it.3. Put it on rocker for at least 15-20 minutes at room temperature.4. Centrifuge at 3500rpm for 15 minutes.Discard the supernatant in acontainer without disturbing the pellet..

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"Protocol1. Take 10ml of blood sample.2. Add 35ml of Tris-EDTA buffer to it.3. Put it on rocker for at least 15-20 minutes at room temperature.4. Centrifuge at 3500rpm for 15 minutes.Discard the supernatant in acontainer without disturbing the pellet.5. Repeat steps till RBC’s are lysed and removed completely. Usually 2washings are enough for the lysis to occur.6. Disrupt the pellet. Add 4.5ml Nuclease buffer, 1.1ml 5M Sodiumperchlorate and 125ul of 10 % SDS. Mix thoroughly with the pipette.7. Rock at RT for 15 minutes.8. Add 2ml of 6M NaCl.9. Shake vigorously for 30s.10.Centrifuge at 4000rpm for 15 minutes.11. Collect the supernatant in a fresh conical tube without disturbing theproteins settled at the bottom after the centrifugation. Add equalvolume of Isopropanol.12.Swirl the tube to precipitate the DNA.13. Fish out the DNA and transfer to a fresh eppendorf tube.14. Wash the collected DNA with 70% ethanol thrice. 15. Dry pellet overnight at RT.16. Re-suspend the pellet in 500ul TE buffer.0 0 17. Incubate at 37 C till the pellet dissolves or at 50 C for 2 hours.0 18.Take a nano drop reading and store at -20 C.*Take 0.5ml of 0.5M EDTA as anti coagulant.* Retain the washed supernatant till the washing is completely done becausethe pellet is very loose and may require re-centrifugation if lost.Buffers1. 1M TrisDissolve 12.11g of Tris base in 80ml of autoclaved dd water. Adjust pH8.0 with HCl, complete volume to 100 ml. Filter, autoclave and store at0 4 C.2. 0.5M EDTADissolve 1.86g of EDTA in 80ml of dd water. Adjust pH to 8.0 with NaoH.Make volume 100ml. 3. 5M Sodium perchlorate Dissolve 61.2g in 80 ml dd water. Bring final volume to 100ml.4. 10% SDSDissolve 5g SDS in 40ml water. Bring final volume to 50ml.5. 5M NaclDissolve 1.46g in 40ml of dd water. Bring final volume to 50ml.6. 6M NaclDissolve 87.66g in 200ml of dd water. Make final volume 250ml.* This is a supersaturated solution of Nacl.7. Tris-EDTA Buffer5ml of 1M tris1ml of 0.5M EDTAMix with 464ml of dd water.Autoclave.8. Nuclease Buffer7.5ml of 5M Nacl24ml of 0.5M EDTA "