Paul.xu: Created page with "1. Take out glycerol stock, ~do not thaw the stock~~, inoculate 20 ml LB//Kan/Chl with the glycerol stock (1 ul) 2. Grow at 37 oC o/n. 3. Add 1 ml of 10,000X Kan..."

2017-07-24T15:09:41Z

Created page with "1. Take out glycerol stock, ~do not thaw the stock~~, inoculate 20 ml LB//Kan/Chl with the glycerol stock (1 ul) 2. Grow at 37 oC o/n. 3. Add 1 ml of 10,000X Kan..."

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1. Take out glycerol stock, ~do not thaw the stock~~, inoculate 20 ml LB//Kan/Chl  with the glycerol stock (1 ul)

2. Grow at 37 oC o/n.

3. Add 1 ml of 10,000X Kan and 1 ml of 10,000X Chl  1 L LB medium

4. Add the overnight culture  into 1 L LB/K/Chl Medium, Grow 37 oC  2~4 hours.

5. When cell density OD600 is ~1, add 0.5 ml of 1 M IPTG to 1 L culture and induce at '''37 oC for 4 hours or 25 oC '''O/N. 

6. Spin down cells. Weigh cell pellet, record weight. Store cell pellet at -20 oC

7. Add 50 ml '''Buffer A'''  to cell pellet, resuspend cell pellet using a spatula

8. Homogenize the cell suspension with a homogenizer or sonicator

9. Incubate the homogenate at 75 oC for 30 min, mix ocasionally, cool on ice for 15 min.

10. Spin at 6000~ 10000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant. 

11. Add 1.5 ml of Ni beads to the supernatant, let KOD to adsorb to Ni bead on a rotary mixer for 30 min. preferably in cold room or fridge

12. Spin down Ni beads at 1000 rpm for 2 min.

13. Add 25 ml  ml '''Ni Washing Buffer.'''

14. Incubate at 4 oC (on ice) for 2 min, mix every 10 sec.

15. Spin down Ni beads.

16. Repeat step 13~15 3 to 5 times, Spin down beads and remove as much washing buffer as possible.

17. Transfer beads to a 2 ml centrifuge tube.

17. Add 0.5 ml '''Buffer B '''to beads''',  '''incubate on ice for 2 min with gentle mixing every 15 sec.

18. Spin genetly for 30 sec using microcentrifuge.

19. Using gel loading tip, collect ~0.5 ml eluted enzyme into a new tube.

20. Repeat step 17 ~ 19.

21. Measure protein concentration using nanodrop, use Buffer B for blanking.

22. Add 0.5 ml of glycerol to fractions with KOD enzyme, add 2 ul of 1 M DTT.

23. Store KOD at -80 oC freezer.

== Buffers ==

'''Buffer A'''

20 mM Tris-HCl, pH 7.9~8.0

500 mM NaCl

0.1 mM EDTA

0.1% Tween 20

0.1% Nonident-P40

 '''Ni Washing Buffer'''

20 mM Tris-HCl, pH 7.9~8.0

800 mM NaCl

10 mM imidazole 

'''Buffer B'''

20 mM Tris-HCl, pH 7.9~8.0

500 mM NaCl

150 mM imidazole 

5% glycerol

[[Category:Protocols]]

KOD Purification - Revision history

Paul.xu: Created page with "1. Take out glycerol stock, ~do not thaw the stock~~, inoculate 20 ml LB//Kan/Chl with the glycerol stock (1 ul) 2. Grow at 37 oC o/n. 3. Add 1 ml of 10,000X Kan..."