Editing T7 DNA Ligase Purification

== Protocol  ==

Transform <strike>Rosetta BL21 (DE3) pLysS</strike> Nico21(DE3)-pLysN with pT7T3-T7 ligase plasmid.<br> 

1. Grow E coli in 1 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 37 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> 

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 T7 ligase. <br> 

14.&nbsp; Measure protein concentration on fractions containing protein. Run SDS-PAGE on protein fractions to check purity.&nbsp; 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,&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. 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).<br> 

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

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,&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> 

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

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

<br>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.

<br>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<br>10 mM MgCl2<br>1 mM ATP<br>10 mM Dithiothreitol<br>pH 7.5 @ 25°C

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

<br>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.

<br>

== 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.<br><br> [[Image:T7 ligase Gel Filtration 2012 12 4.jpg]]<br> 

{| 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, <font color="red">25</font>, 35, 55, <font color="red">70</font>, 100, 130, 250 

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

<br> 

2012-12-17 

ligation QC 

200ng lamdba DNA ,0.5ul T7 ligase 

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

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

[[Category:Protocols]]