Quy trình tách chiết DNA genome từ tế bào prokaryote
Quy trình dùng CTAB
- Grow the microbe culture 48hrs in a peptone broth. Transfer 20ml to a centrifuge tube and spin. Decant the supernatant.
- Resuspend the pellet in 600ul hot CTAB, vortex, and incubate 1 h at 65°C.
- Spin at 15,000 RPM for 10 min. Transfer the upper aqueous phase to a new tube and add 500ul of phenol/chloroform (1:1). Mix.
- Spin at 15,000 RPM for 5 min. Transfer the upper aqueous phase to a new tube and add one more 500ul of phenol/chloroform. Mix
- Add 1ml ethanol, spin at 15,000 RPM for 30 min. at 40C. Spool DNA onto a glass rod
- Dip a rod into 250ul absolute freezing ethanol 70%. Dry naturally for 5-7h
- Resuspend DNA in at least 20ul TE 0.1X buffer.
- Add 20ul RNase (1mg/ml) solution, and incubate 1 h. at 37°C
- Complete resuspension may take several days. Store DNA at 4°C short term, (-) 20 to (-) 80°C long term.
Testing the quality of DNA
- Mix well the DNA sample extraction. Prepare agarose 0.9%. Mix 5ul sample with 1ul ethidium bromate. Run with electrophoresis. Compare with maker.
- Determine the DNA concentration at OD260/280
- The receiving DNA has concentration about 5000ng/ul and the value of OD260/280 is 1.8-1.9, is good quality for PCR running.