Paul.xu at 21:15, 27 December 2012

2012-12-27T21:15:44Z

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== Protocol ==

Transform <strike>Rosetta BL21 (DE3) pLysS</strike> 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.

<strike>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. </strike>

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

<strike>16. Wash Q column with 5 CV of Buffer D. </strike>

<strike>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)</strike> 

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. [[Image:T7 ligase Gel Filtration 2012 12 4.jpg]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 291px; height: 167px;"
|-
| 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

[[Image:T7Ligase 2012 12 5.jpg]]

 

2012-12-17

ligation QC

200ng lamdba DNA ,0.5ul T7 ligase

[[Image:T7 QC 12-17-2012.JPG]] 

{| width="548" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
|-
| digested enzyme
| EcoRV 
| Hind111 
| colspan="3" | Hind111 
| colspan="3" | EcoRV 
|-
| ligate time(min)
| 
| 
| 5 
| 15 
| 30 
| 5 
| 15 
| 30 
|}

[[Category:Protocols]]

T7 DNA Ligase Purification - Revision history

Paul.xu at 21:15, 27 December 2012