Genomic DNA extraction - For PCR

·         Pros: You’ll get genomic DNA quickly from any plant (even with trees).

·         Cons: You need to prepare many reagents.

1.     Meterials

·         Lysis buffer (for 100 mL)

2 M Tris-HCL, pH 7.6	2.5 mL
5 M NaCl	2 mL
0.5 M EDTA, pH 8.0	10 mL
ddw	83 mL

Autoclave for 15 min (121ºC)

20% SDS	2.5 mL
14.26 M BME	70.1 uL

·         TEN buffer

·         Phenol, pH 7.5~8

·         Phenol/chloroform (1:1)

·         Chloroform

·         Isopropanol

·         70%, 100% EtOH

·         TE, pH 8.0

·         10 mg/mL RNase

·         Liquid Nitrogen

·         Pellet pestle (Sigma)

2.     Procedure

1.     Put a 100~200 mg of frozen leaf sample (using Liquid N2) into E-tube and grind by pellet pestle

2.     Put 1.2 mL of lysis buffer and wait for 1~2 hrs at room temperature

3.     Centrifuge for 15 min at 12,000 rpm

4.     Transfer the 1.2 mL of supernatant to 2 mL tube

5.     Add 300 uL of phenol (pH 7.5~8) and shake it for 10 sec. Wait for 2 min at RT

6.     Add 300 uL of chloroform and shake well

7.     Centrifuge for 3 min at 12,000 rpm

8.     Transfer the 1.1 mL of supernatant to 2 mL tube. Repeat (2) to (7) and transfer the 1.0 mL of supernatant to new E-tube

9.     Add same amount of chloroform and shake well

10. Centrifuge for 3 min at 12,000 rpm and transfer the 950 uL of supernatant to 2 mL tube

11. Add same amount of isopropanol and wait for 30 min at RT

12. Centrifuge for 15 min at 12,000 rpm and discard the supernatant. Add 1 mL of 70% EtOH to precipitant and keep it for 10 min in ice bucket. Centrifuge it for 5 min at 6,000 rpm and dry lightly (Caution! Don’t fully dry it. It’ll be very hard to resuspend fully dried sample)

13. Add 500 uL of TEN buffer which contains 50 ug/mL of RNase to precipitant and dissolve it

14. Wait for 30 min at 37ºC

15. Extract with 500 uL of phenol, 500 uL of phenol/chloroform, and 500 uL of chloroform

16. Add 0.1 volume of 3 M NaOAc and precipitate with 2.5 volume of 100% EtOH. Repeat (12) to(15)

17. Dissolve it with 50 uL of TE

3.     Cautions!

·         Sample grinding step is the most important step.

·         4ºC also available for step (3) and (12).

·         Quality is more important than Quality at step (10)

·         You can put at most 400 mg for one tube.

·         You can put RNase at step (2) instead of step (13) if the sample amount is not sufficient.

·         Do not use a Vortex. It can degrade genomic DNA.

·         If you use enlarged pipette tip (you can make it by cutting the end of tip), genomic DNA has less chance for degradation.