KOD Purification
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
[edit]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