Wild-type FL Taq Purification

Transformation

1. Transform BL21 (DE3) pLysS with 1032    pET6XHis-sso7d-wt-Taq plasmid.

2. Grow on LB/Kan/Chl plate o/n.

3.  Pick a few colonies, grow in 15 ml LB/Kan/Chl. Spin down cells to make glycerol stock, Store at -80 oC

Expression

1. Take out glycerol stock and inoculate 200~500 ml LB//Kan/Chl.

2. Grow at 37 oC o/n.

3. Add 1 ml of 10,000X Kan and 1 ml of 10,000X Chl, 20 ml of 20X50 solution to 1 L LB medium to make Autoinduce Medium

4. Split the overnight culture  into 8 L Autoinduce Medium, Grow 37 ^oC  2~4 hours.

5. When cell density OD600 is ~1, add 0.5 ml of 1 M IPTG to 1 L culture and induce at 37 oC O/N. 

5. In the morning, spin down cells, combine. Weigh cell pellet, record weight. Store cell pellet at -20 oC

6. Continue to grow cells (8 L per day) for a few more days until total cell pellet is >100 g.

Purification

1. Add 800 ml Buffer A  to cell pellet, resuspend cell pellet using a spatula

2. Homogenize the cell suspension with a homogenizer.

3. Incubate the homogenate at 75 ^oC for 30 min, mix ocasionally, cool on ice for 15 min.

4. Spin at 6000 rpm for 15 min at 4 ^oC using high speed centrifuge, collect supernatant. 

5. Add cold 400 ml Buffer A to pellet. Homogenize again. 

6. Spin at 6000 rpm for 15 min at 4 ^oC using high speed centrifuge, collect supernatant. 

5. Transfer supernatant to ultracentrifuge tubes.  Cool ultraspeed rotor with dry ice for 5 min.

6. Spin at 25000 rpm for 30 min at 4 ^oC using ultra speed centrifuge, collect clarified supernatant.

7. Load supernatant to 20 ml Ni column in cold room, flow rate ~ 1 ml/min

8. Wash Ni column with 200~500  ml Ni Washing Buffer.

9. Wash Ni column with 50 ml  20 mM Tris-HCl Buffer, pH 8.0

16. Elute Taq enzyme with ~200 ml Buffer B, Collect 5 ml fractions using a fraction collector

17. Measure protein concentration in each fraction. Identify protein peak, combine fractions with OD280 >0.05.

18.  Add 1 volume of cold Dilution Buffer.

19. Load Taq onto a  20~25 ml Q HP column (1.6 X10 or 1.6 X20) using a peristaltic pump

20. Wash column with 100 ml Tris-HCl Buffer, flow rate 2 ml/min

21. Elute Taq using 200 ml of linear gradient of  

A: Q HP Washing Buffer

B: Q HP Elution Buffer  

Flow rate 1.5 or 2 ml/min

22. Run SDS-PAGE gel on the first peak. If there is anothe peak at ~600~800 mM KCl, it could be DNA/RNA peak, discard the DNA/RNA peak.

23. Combine fractions containing Taq.

24. Concentrate Taq using spin column.

25. Freeze-dry Taq in 1 mg per tube

150-ml BioRex 70 (Bio-Rad) ion exchanger column, pre-equilibrated with Buffer C. Collect flow through to see if Taq is bound to the resin. 

Optional: Load supernatant onto Ni column, wash with 20 mM Imidazole in lysis buffer (Buffer B), elute Taq with 300 mM imidazole in Buffer B. Desalt fractions containing Taq using gel filtration column. Combine fractions containing Taq and concentrate by spin concentrator.

22. Wash column with 6 CV of Buffer C, or until no protein or DNA is detected. Collect washes.

23. Elute Taq with Buffer D.

24. Pool fractions containing Taq protein, measure protein concentration. Run SDS gel to check purity.

25. Dilute the partially purified polymerase four-fold with Buffer E (to a final concentration of 50 mM NaCl). Load it into a Q Sepharose FF column. Collect flow through.

26. Wash column with 150 mL Buffer F to wash the column. Check the OD₂₆₀ (it should be less than 0.1) and OD₂₈₀ (it should be less than 0.1).

27. Elute Taq with Buffer G.

28. Collect eluate in 5 to 15 ml per fraction, Taq will come out after 40 mL. Check the OD₂₆₀ and OD₂₈₀ of each fraction. Save all the fractions that have OD₂₈₀ >0.1.

29. Combine the fractions with OD₂₈₀>0.1. Run SDS-PAGE gel to check purity. 

Citraconylation

30. Citraconylate  combined fractions

31. Dialyze citraconylated Taq for 12 h against 3~6 changes of 100 ml  Storage Buffer. The volume will decrease and the protein concentration should be increased to 10-20 mg/ml. 

QC

Make serial dilutions

2/18/17

Name	Dilution	Taq	Dilution Buffer
D1	1:10	Undiluted (~1.64 mg/ml), 1 ul	9 ul
D2	1:100	D1, 1 ul	9 ul
D3	1:1000	D2, 1 ul	9 ul
D4	1:10000	D3, 1 ul	9 ul
D5	1:100000	D4, 1 ul	9 ul

2/19/17

Name	Dilution	Concentration	Taq	Dilution Buffer
D1	1:x	1 ug/ul	dilute to 1 mg/ml	Y ul
D2	1:10 of D1	100 ng/ul	D1, 1 ul	9 ul
D3	1:100 of D1	10ng/ul	D2, 1 ul	9 ul
D4	1:1000 of D1	1 ng/ul	D3, 1 ul	9 ul
D5	1:10000 of D1	0.1 ng/ul	D4, 1 ul	9 ul

PCR Mix without Taq

100 ul 10 uM F

100 ul 10 uM F

Template: 1 ul of 1 mg/ml maxiprep DNA.

400 ul of 10X PCR Buffer with Mg++, 

100 ul 10 mM dNTP

ddH2O to 4 ml

PCR Reaction Mix

#	PCR Mix without Taq	DIluted Taq
1	14.5 ul	0.5 ul D1
2	14.5 ul	0.5 ul D2
3	14.5 ul	0.5 ul D3
4	14.5 ul	0.5 ul D4
5	14.5 ul	0.5 ul D4
6	14.5 ul	0.5 ul Taq + control

PCR Reaction

95 oC 1 min

30 cycles: 95 oC 15  sec, 55 oC 15 sec, 72 oC 30 sec

72 oC 3 min. 

Run on 1% agarose gel after PCR

2/18/17

2/19/17

2/23/2017

Primer DImer QC

Assay protocol Taq_for_Real_Time_PCR

Per reaction:

PCR Mix without Taq & Primers: 13.5 ul

10 uM F Primer: 0.5  ul

10 uM R Primer:  0.5 ul

Taq: 0.5 ul

#	Taq	Assay mix	Note
1	0.5 ul	14.5 ul	50 ug/ml Taq T (Trehalose), freeze-dried, hot start:
2	0.5 ul	14.5 ul	50 ug/ml Taq S (Sucrose), freeze-dried, hot start:
3	0.5 ul	14.5 ul	2/18 Taq, not hot start
4	0.5 ul	14.5 ul	2/1 Taq, not hot start

Observation: looks like hot start Taq do not produce primer dimer (PD)

2/23/2017

Primer DImer QC

Assay protocol Taq_for_Real_Time_PCR

Per reaction:

PCR Mix without Taq & Primers: 13.5 ul

10 uM F Primer: 0.5  ul

10 uM R Primer:  0.5 ul

Taq: 0.5 ul

#	Taq	Assay mix	Note
1	0.5 ul	14.5 ul	50 ug/ml Taq T (Trehalose), freeze-dried, hot start:
2	0.5 ul	14.5 ul	50 ug/ml Taq S (Sucrose), freeze-dried, hot start:
3	0.5 ul	14.5 ul	2/18 Taq, not hot start
4	0.5 ul	14.5 ul	2/1 Taq, not hot start

Observation: looks like hot start Taq do not produce primer dimer (PD)

2/24/2017

Primer DImer QC

Assay protocol Taq_for_Real_Time_PCR

Per reaction:

PCR Mix 1 without Taq & Primers: 13.5 ul

PCR Mix 2 without Taq & Primers with extra template: 13.5 ul

10 uM F Primer: 0.5  ul / 1.0  ul

10 uM R Primer: 0.5 ul / 1.0  ul

Taq: 0.5 ul

#	Taq	Primer F/R	Mix type	Assay mix	Note
1	0.5 ul	0.5 ul	1	13.5 ul	50 ug/ml Taq T (Trehalose), freeze-dried, hot start:
2	0.5 ul	0.5 ul	1	13.5 ul	50 ug/ml Taq S (Sucrose), freeze-dried, hot start:
3	0.5 ul	0.5 ul	1	13.5 ul	2/18 Taq, not hot start
4	0.5 ul	0.5 ul	1	13.5 ul	2/1 Taq, not hot start
5	0.5 ul	1.0 ul	1	13.5 ul	50 ug/ml Taq T (Trehalose), freeze-dried, hot start:
6	0.5 ul	1.0 ul	1	13.5 ul	50 ug/ml Taq S (Sucrose), freeze-dried, hot start:
7	0.5 ul	1.0 ul	1	13.5 ul	2/18 Taq, not hot start
8	0.5 ul	1.0 ul	1	13.5 ul	2/1 Taq, not hot start
9	0.5 ul	0.5 ul	2	13.5 ul	50 ug/ml Taq T (Trehalose), freeze-dried, hot start:
10	0.5 ul	0.5 ul	2	13.5 ul	50 ug/ml Taq S (Sucrose), freeze-dried, hot start:
11	0.5 ul	0.5 ul	2	13.5 ul	2/18 Taq, not hot start
12	0.5 ul	0.5 ul	2	13.5 ul	2/1 Taq, not hot start
13	0.5 ul	1.0 ul	2	13.5 ul	50 ug/ml Taq T (Trehalose), freeze-dried, hot start:
14	0.5 ul	1.0 ul	2	13.5 ul	50 ug/ml Taq S (Sucrose), freeze-dried, hot start:
15	0.5 ul	1.0 ul	2	13.5 ul	2/18 Taq, not hot start
16	0.5 ul	1.0 ul	2	13.5 ul	2/1 Taq, not hot start

Observation: looks like hot start Taq do not produce primer dimer (PD)

Conclusion

Old Batch 2/1/17: D3 dilution is good enough

New Batch 2/18/17: D2 or D3 is good enough:

5 ng~50 ng per reaction is good enough 

Regeneration of Ni column

1. Wash Ni column with 5 CV of water.

2. Wash column with 3 CV of 100 mM EDTA, 500 mM NaCl.

3. Wash the column with 5 CV of H2O.

4. Recharge the column with 1 column volume of 100 mM NiSO4, or NiCl2.

5. Wash the column with 10 column volumes of H2O.

6. Equilibrate with 3 volumes of a suitable buffer (e.g., Buffer A).

Buffer A

20 mM Tris-HCl, pH 7.9~8.0

500 mM NaCl

0.1 mM EDTA

0.1% Tween 20

0.1% Nonident-P40

Ni Washing Buffer

20 mM Tris-HCl, pH 7.9~8.0

800 mM NaCl

10 mM imidazole 

Tris-HCl Buffer 

20 mM Tris-HCl, pH 7.9~8.0

Buffer B (Low Salt)

20 mM Tris-HCl, pH 7.9~8.0

100 mM NaCl

150 mM imidazole 

5% glycerol

Dilution Buffer

20 mM Tris-HCl, pH 7.9~8.0, optional: 5% glycerol

Q HP Washing Buffer

20 mM Tris-HCl, pH 7.9~8.0

50 mM KCl

5% Trehalose

Q HP Elution Buffer

20 mM Tris-HCl, pH 7.9~8.0

1 M KCl

5% Trehalose

Buffer E

20 mM HEPES (pH 8.3) or Tris-HCl, pH 8.0 

Buffer F

20 mM HEPES (pH 8.0) or Tris-HCl, pH 7.9 

50 mM KCl. 

Buffer G

20 mM HEPES or Tris-HCl, pH 7.9 or pH 8.0

500 mM KCl. 

Storage Buffer

20 mM HEPES, pH 7.9

100 mM KCl

0.1 mM EDTA

1 mM dithiothreitol

50% glycerol.

0.5 ul