CTAB method

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1. Turn on the water bath (65°C)

2. Grind plant samples as fast as possible and put into 2ml tube (store -70°C before use)

3. Prepare the pre-heated CTAB buffer with 2% PVPP and 2% β-mercaptoethanol(750ul/sample)

4. Incubate samples with CTAB buffer at 65°C water bath for 30min (gently inversion every 10 min)

5. Put PCI (Phenol:Chloroform:Isoamylalcohol = 25:24:1) into the sample tube and mix well (voltex)

6. Gently inversion for 30 min using shaking incubator

7. after that, spin down sample tubes at 12,000 rpm for 30 min (twice, 25°C)

8. Keep the supernatant (~750ul/tube) and put into new 1.5ml tube

9. Then, put 5M NaCl(200ul) and cold Iso-propanol(600ul) into the each tube

10. Gently mix and incubate samples at -20°C for 30 min

11. Spin down samples at 12,000 rpm for 10 min

12. put off the supernatant and add 70% alcohol into tube (you can see the white pellet!)

13. Spin down samples at 12,000 rpm for 10 min and pour off the alcohol and repeat centrifuge step

14. Dry DNA pellet

15. Add 100~200ul ddH20 with RNAse(RNase 1ul/ddH2O 100ul)

Material information

2ml 기준

Chemical Volume
CTAB buffer 800 ul
PCI 800 ul
BME 16 ul
Supernatant 700 ul - 750 ul
5M NaCl 200 ul
Iso-propanol 600 ul
EtOH (70%) 500 ul
TE buffer 50 ul
RNase A 1 ul
비고 추후 4 tube 합침 : 200ul

50ml 기준(20X)

Chemical Volume
CTAB buffer 15ml
PCI 15ml
BME 320ul
Supernatant 12ml
5M NaCl 3ml
Iso-propanol 9ml
EtOH (70%) 10ml
TE buffer 1ml
RNase A 20ul