T7 DNA Ligase Purification

Protocol

Transform Rosetta BL21 (DE3) pLysS Nico21(DE3)-pLysN with pT7T3-T7 ligase plasmid.

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 for o/n at 37 oC.

4. Spin down cells, resuspend in  Buffer A (50 mM Tris-HCl, pH 7.5,  10 mM imidazole, 10% glycerol,  500 mM NaCl,  1 mM beta-ME).

Suspension 5 ml per g pellet. 30g --> 150 ml Buffer A.

5.  Lyse cells by freezing and thawing then  homogenization.

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

7. Resuspend pellet in Buffer A (5 ml buffer/g pellet). Homogenize and spin again. Pool supernatant.

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

9. Load  clear supernatant to a 20~30  ml Ni column.

10. Wash with 400 ml Washing Buffer (50 mM Tris-HCl, pH 7.5,  500 mM NaCl, 1 mM beta-ME) then 100 ml of Buffer A.

11. Elute with Buffer B (300 mM imidazole in Buffer A). Collect 5 ml fractions. Concentrate fractions containing protein using 2 Pierce 9KDa spin column. (to ~5 ml each)

12. Load 5 ml  to a Tosoh gel filtration BioRad Bio-Sil TSK250 column, elute with Buffer C (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.1 mM EDTA, 5 mM beta-ME)

13. Collect 2.5 ml fractions. Determine protein concentration in  fractions containing T7 ligase.

14.  Measure protein concentration on fractions containing protein. Run SDS-PAGE on protein fractions to check purity.  Protein purity should be at least 98%. Pool T7 DNA ligase fractions. Concentrate using two 9KDa spin column.

14. Dilute fractions containing T7 ligase 1 fold with Buffer D (50 mM Tris-HCl, pH 7.5,  0.1 mM EDTA, 5 mM beta-ME) to  reduce NaCl concentration to less than 50 mM.

15. Load to a Q-Sepharose column (10 ml) pre-equilibrated in Buffer D.

16. Wash Q column with 5 CV of Buffer D.

17. Elute protein with linear gradient to Buffer E (50 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 1 mM EDTA, 5 mM beta-ME)

18. 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).

19. Take 2 ul and dilute to 2 ug/ul with Storage Buffer. Check enzyme activity using Fast T7 DNA Ligase Buffer.

Digest 500 ng plasmid DNA with enzymes such as EcoR1 + Xho1 to cut out insert and vector. Combine two gel slices and gel purify. Add T7 DNA ligase and T7 DNA ligase buffer, (66 mM Tris-HCl,  10 mM MgCl₂, 1 mM ATP, 1 mM DTT, 7.5 % PEG 6000, pH 7.6), ligate for 30 min. Run gel to see if two DNA fragments become one fragment after 0.5 hour ligation.

Specific activity should be 3,000,000 U/mg

Product for sale: dilute to 1 mg/ml (3,000,000 U/ml, 3000U/ul) with Storage Buffer.

Fast T7 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5 % PEG 6000, pH 7.6)

Make 5 X Buffer:

75g PEG 6000

50 ml of 1M Tris-HCl, pH 7.6

10 ml of 1 M MgCl2

18,3 MOhm ultrapure water to 200 ml

Autoclave, make sure buffer is not evaporated (measure the volume with autoclaved 100 ml measuring cylinder, add autoclave water to ~ 188.5ml if evaporated).

Add 2 ml of 0.5 M ATP, 0.5 ml of 2 M DTT.

If it is very viscous, dilute the buffer with water (add 50 ml autoclaved 18,3 MOhm ultrapure water) to make 4 X buffer.

Slow T7 DNA Ligase Buffer:

50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM Dithiothreitol
pH 7.5 @ 25°C

Run gel to see if two fragments become one fragment after 1 hour ligation.

20. Create a record in protein database. Write detail description on every step.

21. Very Important: Do not dilute protein!!!.

Aliquote undiluted protein: 1 ml to 1.5 ml microcentrifuge tube. Label them and store undiluted protein in a freezer box at -80 oC.

Method Validation

8 L culture: induction with 0.3 mM IPTG, 37 oC, O/n. 30 g wet cells.

Lyse with 2 X 150 ml buffer A. Wash Ni beads with 400 ml Buffer A.

Fraction	Protein, mg/ml	Vol, ml
34	0.19	12
35	1.95	12
36	1.8	12
37	0.77	12
38	0.36	12
39	0.1	12

Take 10 ul to run RunBlue gel, marker is Fermentas PageRuler Plus protein ladder

10, 15, 25, 35, 55, 70, 100, 130, 250

2012-12-17

ligation QC

200ng lamdba DNA ,0.5ul T7 ligase

	1	2	3	4	5	6	7	8
digested enzyme	EcoRV	Hind111	Hind111			EcoRV
ligate time(min)			5	15	30	5	15	30