T4 DNA Ligase 20121228

Gel filtration:

Protocol

1. Grow E coli in 1 L LB/Kan/Chl at 37 oC until OD280 ~ 0.6-0.7. Total is 8 L

3. Induce with 0.3 mM IPTG o/n at 37 oC.

4. Spin down cells, resuspend in Buffer A (20 mM Tris-HCl, pH 7.5, 10 mM imidazole, 10% glycerol, 500 mM NaCl, 5 mM beta-ME).

Suspend 10 ml per g pellet. 30g --> 300 ml Buffer A.

5. Lyse cells by freezing and thawing then homogenization briefly.

6. Spin at 12000 rpm for 15 min, 4 oC.

7. Resuspend pellet in Buffer A (10 ml buffer A/g pellet). Homogenize and spin again. Pool supernatant.

8. Spin at 25000 rpm for 20 min, 4 oC.

9. Load clear supernatant to a 45 ml Ni column.

10. Wash with 500 ml 'Washing Buffer'I (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Triton X-100 or NP-40, 5 mM beta-ME), then 300 ml Wash Buffer II (20 mM Tris-HCl, pH 7.5, 10 mM imidazole, 500 mM NaCl, 5 mM beta-ME). You can use peristaltic pump to wash the Ni column.

11. Elute with Buffer B (20 mM Tris-HCl, pH 7.5, 250 mM imidazole, 200 mM NaCl, 5 mM beta-ME). Collect 5 or 10 ml fractions. Discard the 1 CV (45 ml if Ni resin column is 45 ml).

12. Measure protein conc using Buffer B as blank. Combine fractions containing protein.

13. Equilibrate a 5 ml HiTrap Q FF column with Buffer B. Load protein solution to the column. T4 DNA ligase will not find and flow through should be collected. Flush out unbound protein using addition 10 ml of Buffer B.

After use, HiTrap Q FF column should be regenerated using 1.5M NaCl/0.1N NaOH, wash column with water and store the column in 20% ethanol.

14. Concentrate T4 DNA ligase using 2 Pierce 9k spin columns to 7~ 10 ml.

15. Load 3~ 4.5 ml to a Tosoh gel filtration BioRad Bio-Sil TSK250 column, elute with Buffer C (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM beta-ME).

Run > 2 times if you have more than 5 ml concentrated protein solution.

16. Collect 2.5 ml fractions. Determine protein conc in peak fractions. Run SDS-PAGE gel to check purity. Pool fractions containing T4 DNA ligase.

24. Concentrate protein solution using two Pierce 9k spin columns. After concentration add DTT from 1 M or 2 M stock to protein solution to adjust final concentration of 1 mM. Dialyse protein solution vs Storage Buffer (20 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA, 50% Glycerol, pH 7.5).

25. Take 2 ul and dilute to 2 ug/ul with Storage Buffer. Check enzyme activity using Fast T4 DNA Ligase Buffer