Editing T3 DNA Ligase Purification

== Protocol  ==

Transform <strike>Rosetta BL21 (DE3) pLysS</strike> Nico21(DE3)-pLysN with pT7T3-T3 DNA ligase plasmid.<br> 

1. Grow E coli in 8 L&nbsp; LB/Kan/Chl at 37 oC&nbsp; until OD280 ~ 0.6-0.7. Total is 8 L<br> 

3. Induce with&nbsp; 0.3 mM IPTG for o/n at 25 oC. <br> 

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

Use used 50% glycerol. Glycerol is optional. 

Suspension 5 ml per g pellet. 30g --&gt; 150 ml Buffer A. 

5.&nbsp; Lyse cells by freezing and thawing then&nbsp; homogenization.<br> 

6. Spin&nbsp; 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&nbsp; clear supernatant to a 20~30&nbsp; ml Ni column. 

10. Wash with 400 ml '''Washing Buffer''' (50 mM Tris-HCl, pH 7.5,&nbsp; 500 mM NaCl, 1 mM beta-ME) then 100 ml of '''Buffer A. '''<br> 

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)<br> 

12. Load 5 ml&nbsp; 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)<br> 

13. Collect 2.5 ml fractions. Determine protein concentration in&nbsp; fractions containing T3 DNA ligase. Run SDS-PAGE gel to check purity. <br> 

14. Pool T3 DNA ligase fractions. Concentrate using 2&nbsp; 9KDa spin column. 

<strike>14. Dilute fractions containing T3 ligase 1 fold with '''Buffer D''' (50 mM Tris-HCl, pH 7.5,&nbsp; 0.1 mM EDTA, 5 mM beta-ME) to&nbsp; reduce NaCl concentration to less than 50 mM.<br></strike> 

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

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

18. Dialyse protein&nbsp; vs storage buffer (20 mM Tris-HCl, 50 mM KCl or NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4).<br> 

19. 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 T3 DNA ligase and T3 DNA ligase buffer, (66 mM Tris-HCl,&nbsp; 10 mM MgCl<sub>2</sub>, 1 mM ATP, 1 mM DTT, 7.5&nbsp;% 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. 

<br> 

'''Make 5 X Buffer:''' 

75g PEG 6000 

50 ml of 1M Tris-HCl, pH 7.6 

10 ml of 1 M MgCl<sub>2 </sub> 

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. 

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

21. Store undiluted protein at -80 oC.<br> 

[[Image:T3_ligase_Purification_Ni_resin_1_2013.jpg]]<br> 

[[Image:T3 Ligase purification anion exchange 1 2013.jpg]] 

== Method Validation  ==

<br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 291px; height: 167px;"
|-
| Fraction 
| Protein, mg/ml 
| Vol, ml
|-
| 34 
| <br> 
| <br>
|-
| 35 
| <br> 
| <br>
|-
| 36 
| <br> 
| <br>
|-
| 37 
| <br> 
| <br>
|-
| 38 
| <br> 
| <br>
|-
| 39 
| <br> 
| <br>
|}

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

[[Category:Protocols]]