Paul.xu at 16:59, 9 January 2013

2013-01-09T16:59:54Z

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== Protocol ==

Transform <strike>Rosetta BL21 (DE3) pLysS</strike> Nico21(DE3)-pLysN with pT7T3-T4 DNA 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  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,  1 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, 1 mM beta-ME), then 100 ml '''Wash Buffer II '''(20 mM Tris-HCl, pH 7.5, 10 mM imidazole,  10% Glycerol,  1 M NaCl, 1 mM beta-ME). 50 ml '''Wash Buffer III''' (20 mM Tris-HCl, pH 7.5, 10 mM imidazole, 10% Glycerol, 500 mM NaCl, 1 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, 1 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).<strike> </strike>

Run > 2 times if <strike></strike>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. 

'''If protein is not pure enough, do step 17 to 23:'''

17. Equilibrate a '''45 ml Q Sepharose FF XK26''' column with 100 ml  '''Buffer D'''  (20 mM Tris-HCl, pH 8.0,  '''50 mM''' NaCl, 1 mM beta-M). 

18. Dilute protein solution with 0.5 to 1 volume of  '''Buffer E '''(20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM beta-M).  Load the diluted solution onto the Q column. Collect FT after 1 CV (45 ml), check protein concentration using buffer C as blank.  Protein conc should be very low in FT. 

19. Wash Q column with 100 ml (2 CV) of Buffer D. 

20. Wash Q column with 50 ml (1 CV) of '''Buffer F''' (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM beta-M). 

21. Connect XK column to FPLC. Elute protein with 200 ml linear gradient of '''Buffer D'''  to  '''Buffer F '''(50 mM Tris-HCl, pH 7.5, 500 mM NaCl,  1 mM beta-ME), flow rate 2 ml per min. Collect 4 ml per fraction. 

22. Measure protein concentration on fractions containing protein.  Run SDS-PAGE on protein fractions. Protein purity should be at least 98%. 

23. Pool fractions containing pure protein. Measure protein concentration. 

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''' 

 

Specific activity: should be 300,000 U/mg. 

 

Product for sale: dilute to 2 mg/ml (600,000 U/ml, 600U/ul) with '''Storage Buffer'''.

 '''Fast T4 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 T4 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.

 

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

27. '''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. 

 

28. Regenerate Q column using 100 ml CIP Solution (1.5 M NaCl, 0.1 N NaOH). Wash with 150 ml water and the flush with 60 ml 20% ethanol if the column will not be used in a few days.

 

== Method Validation ==

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

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

{| cellspacing="1" cellpadding="1" border="1" style="width: 291px; height: 167px;"
|-
| Fraction
| Protein, mg/ml
| Vol, ml
|-
| 54 
| 0.1 
| 7.5 
|-
| 55 
| 0.55 
| 7.5 
|-
| 56 
| 1.4 
| 7.5 
|-
| 57 
| 1.6 
| 7.5 
|-
| 58 
| 1.14 
| 7.5 
|-
| 59 
| 0.86 
| 7.5 
|-
| 60 
| 0.5 
| 7.5 
|-
| 61 
| 0.28 
| 7.5 
|}

Take 10 ul to run RunBlue gel, marker is Invitrogen protein ladder SeeBlue® Plus2 

M: 198, 98 (orange), 62, 49, 38, 28, 17 (pink), 14, 6, 3 

MW: T4 ligase is  55292.65 

PI= 6.14 

Gel filtration, 2.5 ml/fraction

[[Image:T4 ligase gel filtration purification 2012 12 08.jpg]]

Take 10 ul to run gel

[[Image:T4 ligase purification 2012 12 9.jpg]] 

 

2013/1/3 

T4 QC 

200ng lambda DNA

[[Image:T4 ligase QC 1-3-2013.JPG]][[Image:T4 liagse QC control 1-3-2013.JPG]]

{| width="589" cellspacing="1" cellpadding="1" border="1"
|-
| lane
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
|-
| 
| colspan="4" | Hind111
| colspan="4" |   EcoRV
| Hind111 digested DNA
| EcoRV digested DNA
|-
| Ligation   time min
| 5
| 
| 30
| 60
| 5
| 15
| 30
| 60
| Congtrol
| control
|}

 

2013/1/7--2013/1/9 

Ligation QC

1597-pT7A3-6XHis.GST 

[[Category:Protocols]]

T4 DNA Ligase Purification - Revision history

Paul.xu at 16:59, 9 January 2013