Genomic DNA extraction – using a CTAB buffer

·         You’ll get a relatively clean genomic DNA from a sample with phenolic compounds or polysaccharides using this method.

1.     Materials

·         2x CTAB buffer

2% (w/v) CTAB (cetyltrimethylammonium bromide) (Spectrum Chemical Mfg. Corp., Sigma)

100 mM Tris-HCl, pH 8.0

20 mM EDTA, pH 8.0

1.4 M NaCl

1% polyvinylpyrrolidine (mw 40,000) (Sigma)

·         CTAB precipitation buffer

1% (w/v) CTAB

50 mM Tris-HCl, pH 8.0

10 mM EDTA, pH 8.0

·         1x CTAB

Dilute 2x CTAB

·         TE

·         CsCl solution

10 g CsCl/10 mL TE

·         10 mg/mL EtBr solution

·         20x SSC saturated 2-propanol

Using a upper part when you mix 100 mL of 2-propanol and 50 mL of 20x SSC

·         Chloroform

·         70%, 100% EtOH

·         20x SSC

2.     Procedure

a.      Preheat 2x CTAB and 1x CTAB in 65ºC water bath. Wash a plant sample with ddw and remove an excessive water using a paper towl.

b.     Freeze 10 g of fresh tissue with liquid N2 and grind with pestle and mortar.

c.      Transfer the powder into centrifuge tube.

d.     Add 1 mL of preheated 2x CTAB buffer per gram of powder and mix them.

e.      Add 4 mL of preheated 1x CTAB buffer per gram of powder and mix them. And keep it in 65ºC water bath for 5 min.

f.       Add 10 mL of chloroform and mix them well.

g.     Centrifuge it for 5 min at 12,000 rpm, 4ºC.

h.     Transfer the supernatant to new tube.

i.       Add 2 volume of CTAB precipitation buffer and mix them briefly.

j.       Keep it for 30 min at RT.

k.     Centrifuge it for 5 min at 12,000 rpm, RT

l.       Discard the supernatant and dry the precipitant briefly.

m.   Add 4 mL of CsCl solution per 5 g of sample and dissolve it (it may take more than 1hr).

n.     Add 0.1 mL of EtBr and mix them briefly.

o.     Transfer the solution into ultracentrifuge tube and seal it.

p.     Ultracentrifuge it for 6hrs at 65,000 rpm or overnight at 55,000 rpm at 15ºC.

q.     Pick up the DNA band using a syringe and transfer it to E-tube.

r.       Add 0.5 mL of 20x SSC saturated 2-propanol and mix them. Transfer the clear bottom part.

s.      If the DNA band is strong, add 2x volume of ddw and 8x volume of cold 95% EtOH into (p) and mix them.

t.       After the ultracentrifuge, wash the precipitant with cold 70% EtOH.

u.     Discard supernatant and dry the precipitant (DNA).

v.     Add 0.5 mL of TE buffer and dissolve it.

w.   You can dialyze twice times against 1 liters TE buffer per hour to remove remained CsCl instead of EtOH precipitation.

x.     Collect DNA into E-tube using a membrane bag.

Plant genomic DNA extraction (large scale) – Using a Proteinase K buffer

·         You’ll get around 50 ug of genomic DNA from 1 g of plant tissue sample.

·         Expensive but effective

·         Quality is enough for a genomic library preparation.

1.     Materials

·         Proteinase K buffer

0.2 M Tris-HCl, pH 8.0

0.1 M EDTA, pH 8.0

1% sarkosyl (Sigma)

100 ug/mL proteinase K (Sigma)

·         70%, 100% EtOH

·         3 M Sodium acetate, pH 4.8

·         CsCl

·         10 mg/mL Ethidium bromide (EtBr)

·         Ddw-saturated 1-butanol

·         TE

·         Dialysis tube

·         Quick-seal tube (Beckman)

2.     Procedure

a.      Add 0.5~1.5 g of plant tissue with 3 mL of proteinase K buffer into a mortar and grind them.

b.     Transfer it to 15 mL conical tube and stay at 45~50ºC for 1 hour

c.      Centrifuge it for 10 min at 3,000 rpm

d.     Transfer the supernatant to a new tube and adjust the volume to 3 mL using a proteinase K buffer

e.      Add 6 mL of EtOH and mix. Add 300 uL of 3 M sodium acetate, pH 4.8 and shake it

f.       Centrifuge it for 15 min at 10,000 rpm (SS-34 rotor) and discard its supernatant. Air drying the precipitant.

g.     Dissolve the dried precipitant in 4 mL of TE. If the precipitant seems to have many contaminants, dissolve the precipitant in 3mL of TE and repeat (e) to (g).

h.     Mix it with 4.5g CsCl and 400 uL of EtBr (10 mg/mL).

i.       Centrifuge it for 16~20 hrs at 53,000 rpm (VTi 65 rotor), 20ºC. If you have VTi 80 rotor, you can centrifuge it for 12~16 hrs at 55,000 rpm or 8 hrs at 73,000 rpm.

j.       Transfer the genomic DNA band to E-tube and add same amount of ddw-saturated 1-butanol. Mix well and keep for 2 min.

k.     Centrifuge it for 3 min at 1,500x g (RT) and transfer the water layer to new E-tube. Repeat for 4~6 times until the pink color from EtBr is disappeared.

l.       Dialyze overnight against 2 liters TE buffer to remove remained CsCl.

Genomic DNA extraction - Fast extraction method

·         This method is good for organ with highly accumulated phenolic compounds or seedlings.

1.     Materials

·         Homogenization buffer

1 M NaCl

0.2 M sucrose

0.01 M EDTA

0.03 M Tris-HCl, pH 8.0

·         Lysis buffer

0.25 M EDTA

2.5% (w/v) SDS

0.5 M Tris-HCl, pH 9.2

·         Grind buffer (prepare just before use)

4 homogenization buffer + 1 lysis buffer

·         3 M Potassium acetate, pH 4.7

·         70%, 100% EtOH

·         10 mg/mL RNase (Sigma)

·         Pellet pestle (Sigma)

2.     Procedure

a.      Freeze 0.25 g of plant tissue samples in liquid N2, put it to E-tube, and grind with pellet pestle.

b.     Add 400 uL of grind buffer and slightly disturb it at 65ºC (prevent to make any bubbles).

c.      Keep it at 65ºC for 30 min.

d.     Add 13.3 uL of 3 M potassium acetate, pH 4.7

e.      Keep it on ice for 30 min.

f.       Centrifuge it for 10 min at 4ºC, 12,000 rpm and transfer the supernatant to E-tube.

g.     Add 700 uL of 100% EtOH and keep it for 2 min at room temperature (RT). Centrifuge it for 10 min at 4ºC, 12,000 rpm.

h.     Discard supernatant. Wash the precipitant with cold 70% EtOH and centrifuge it for 2 min at 4ºC, 12,000 rpm.

i.       Discard supernatant and dry the precipitant. Dissolve the dried precipitant with 40 uL of ddw and 1uL of DNase-free RNase (10 mg/mL).

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.

EASY-nLC 1000 has Leaks!!!

Proteomics 연구에 사용중인 Thermo의 EASY-nLC 1000에서 leak가 발생했다.

발견하게된 계기는 우선 solvent B (80% ACN, 0.5% acetic acid) 에서 기포가 없애도 없애도 생기길래, 그전에도 문제가 되었었던 check valve를 교체해 봤었다.

교체한 check valve: IDEX CV-3315 ($98.78)

하지만 증상은 나아지질 않았고, 급기야 sample loading중에 error message를 뿜어냈다.

그래서 pump A와 B에 대해서 leak test를 해보니, 역시나 pump B에 문제가 있음을 알 수 있었다.

pump B가 980bar 까지 못가고 있다!!

이런 적이 없었으므로 (이제 설치한지 일년 갓 지났다), 재빨리 Thermo에 연락을 해보니, 아마도 rotor seal을 갈아야 할 것 같다고 했다.
아... 이제 나의 솜씨를 보여줄 때인 건가?