Chemical Modification of Taq in TMBE
Protocols
Purification of Taq
[edit]Grow Taq-plasmid transformed JM109 in 500 ml TB/amp O/N at 37 oC
Split into 10 L of 1 L of TB/amp, grow to OD600=0.7
Induce with 0.1mM IPTG.
Grow for 26 hours
Harvest cells by centrifugation at 3000×g for 15 min.
Freeze cell pellets
Thaw pellet and resuspend in 25mM Tris–HCl (pH 7.5), 10mM EDTA, 0.2M ammonium sulphate, 50 mg/mL lysozyme
Incubate at room temperature for 15 min
Incubate at 75 ◦C for 1 h toprecipitate heat-sensitive proteins.
Spin at 36,000×g for 30 min.
Add Polyethyleneimine to the clarified cell lysate to a final concentration of 0.1% (v/v)
incubate the mixture on ice with agitation for 1 h to precipitate E. coli host nucleic acids.
Spin at 36,000×g for 30 min to precipitate nucleic acids
Load the supernatant onto a 50mL Phenyl-Sepharose® high substitutionfast flow column (Amersham Biosciences) equilibrated with 25mM Tris–HCl (pH 7.5), 1mM EDTA, 0.2M ammonium sulphate.
Wash column with 3 column volumes of 25 mM Tris–HCl (pH 7.5), 1mM EDTA.
Elute Taq polymerasen with a 0–4 M gradient of urea over 9 column volumes.
Pool fractions containing Taq polymerase.
Dialyse to remove traces of urea
Concentrate using a Vivaflow 50 system in 25mM Tris–HCl (pH 7.5), 1mM EDTA
Store enzyme at 4 ◦C.
Chemical Treatment
[edit]Concentrate Taq polymerase in 25 mM Tris–HCl (pH 9.0), containing either 100mM potassium chloride, 2% (w/v) sucrose, or neither additive using spin-columns (Millipore, Microcon 50,000 MWCO), to 1 ml at a concentrate of (8.75U/ul).
Dry the enzyme by rotary evaporation (Savant AES2010 SpeedVac, Thermo Life Sciences) at room temperature until a glassy powder is formed
Add Citraconic anhydride (50 uL) and tert-butyl methyl ether (t-BME, 950uL) to the dried Taq polymerase samples
Incubate overnight at either room temperature or 50 ◦C with continuous agitation
Spin gently
Remove non-aqueous phase by pipetting.
Wash the suspension by addition of anhydrous t-BME to remove any excess reagent and its corresponding acid from the preparation.
Remove remaining solvent under vacuum in the Savant Speedvac.
Resuspend modified Taq polymerase in 1mL water since the buffering salts is still present in the preparation.
Store the enzyme at 4 ◦C (at a theoretical concentration of 8.75U/ul before
modification).
Taq polymerase dried in the presence of either potassium chloride or sucrose was reversibly modified at room temperature and 50 ◦C as shown by the absence of a PCR product unless the enzyme had first been subjected to a 15 min pre-incubation at 95 ◦C. Where the enzyme had been dried in the absence of any additives, the modification was incomplete when the modification was carried out at room temperature, but resulted in denaturation when the modification was carried at 50 ◦C.
When Taq is dried in the presence
of both additives, the enzyme modified at 50 ◦C is more
stable than that modified at room temperature.
1 The DNA polymerase from Thermus aquaticus (50,000 units) is vacuum-dried in the presence of 2% sucrose (lyoprotectant) in double-distilled and de-ionised water., or dried or freeze-dried in the presence of 10 mM Tris HCI at pH 9.2 The resulting amorphous powder is then added to 5 mL anhydrous t-MBE, to which an excess of citraconic anhydride is added to modify the lysine groups in question (5% (v/v)). The solvent is then maintained at 37° C. for 5 hours. At this stage the powder is washed 4 times with 10 mL hexane to remove any remaining contaminating modification reagent. Finally, the powder is stored as is at −20° C., 4° C. or dissolved in aqueous solution, or a storage buffer (20 mM Tris-HCI; 100 mM potassium chloride; 0.1 mM ethylenediaminetetraacetic acid; 1 mM dithiothreitol; 0.5% (v/v) Tween 20; 0.5% (v/v) Nonidet P40; 50% glycerol; pH 9.2) to the desired concentration (usually 5 units/μL) and stored at −20° C.
2. The DNA polymerase from Thermus aquaticus (50,000 units) is dried or freeze-dried in the presence of 1 M Tris HCI at pH 9.2. The resulting amorphous powder is then added to 5 mL anhydrous ethyl acetate, to which an excess of citraconic anhydride is added to modify the lysine groups in question (5% (v/v)). The solvent is then maintained at 50° C. for 5 hours. At this stage the powder is washed 4 times with 10 mL hexane to remove any remaining contaminating modification reagent. Finally, the powder is stored as is at −20° C., 4° C. or dissolved in aqueous solution, or a storage buffer (20 mM Tris-HCI; 100 mM potassium chloride; 0.1 mM ethylenediaminetetraacetic acid; 1 mM dithiothreitol; 0.5% (v/v) Tween 20; 0.5% (v/v) Nonidet P40; 50% glycerol; pH 9.2) to the desired concentration (usually 5 units/μL) and stored at −20° C.
3. The DNA polymerase from Thermus aquaticus (50,000 units) is dried or freeze-dried in the presence of 10 mM Tris HCI at pH 9.2. The resulting amorphous powder is then added to 5 mL anhydrous methyl ethyl ketone, to which an excess of citraconic anhydride is added to modify the lysine groups in question (1% (v/v)). The solvent is then maintained at 50° C. for 5 hours. At this stage the powder is washed 4 times with 10 ml hexane to remove any remaining contaminating modification reagent. Finally, the powder is stored as is at −20° C., 4° C. or dissolved in aqueous solution, or a storage buffer (20 mM Tris-HCI; 100 mM potassium chloride; 0.1 mM ethylenediaminetetraacetic acid; 1 mM dithiothreitol; 0.5% (v/v) Tween 20; 0.5% (v/v) Nonidet P40; 50% glycerol; pH 9.2) to the desired concentration (usually 5 units/μL) and stored at −20° C.
4. The DNA polymerase from Thermus aquaticus (50,000 units) is dried or freeze-dried in the presence of 10 mM Tris HCI at pH 9.2. The resulting amorphous powder is then added to 5 mL anhydrous carbon tetrachloride, to which an excess of citraconic anhydride is added to modify the lysine groups in question (1% (v/v)). The solvent is then maintained at 37° C. for 5 hours. At this stage the powder is washed 4 times with 10 ml hexane to remove any remaining contaminating modification reagent. Finally, the powder is stored as is at −20° C., 4° C. or dissolved in aqueous solution, or a storage buffer (20 mM Tris-HCI; 100 mM potassium chloride; 0.1 mM ethylenediaminetetraacetic acid; 1 mM dithiothreitol; 0.5% (v/v) Tween 20; 0.5% (v/v) Nonidet P40; 50% glycerol; pH 9.2) to the desired concentration (usually 5 units/μL) and stored at −20° C.
Dissolve Taq in buffered aqueous solution (20 mM Tris (pH 8.0); 100 mM KCI; 0.1 mM EDTA; 1 mM DTT; 0.5% Tween 20; 0.5% Nonidet P40; 50% glycerol), before use diluted to 5U/μl before use. Reaction buffer: 10 mM Tris pH 8.3, 50 mM KCI. Activation step (if used) at 95° C. for 15 minutes. The PCR consists of initial denaturation at 94° C. for 2 minutes (1 cycle); denaturation at 94° C. for 20 seconds, annealing at 60° C. for 30 seconds, and extension at 72° C. for 30 seconds (30 cycles); final extension at 72° C. for 5 minutes. Human DNA template concentration was (100 ng/μl); dNTPs (0.2 mM each), 1.5 mM MgCl2; Primer 1 (5′-ATT TGC GGT GGA CGA TGG AG-3′) and Primer 2 (5′-AGA GAT GGC CAC GGC TGC TT-3′) at 250ng each per reaction.