Paul.xu at 13:23, 8 June 2016

2016-06-08T13:23:55Z

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4.8.Medium

4.8.1

2 x YT medium Per liter: Tryptone 16g Yeast extract 10g NaCl 5g Adjust pH to 7.0 with 5N NaOH. Sterilize by autoclaving.

4.8.2

LB medium Per liter: Tryptone 10g Yeast extract 5g NaCl 10g Adjust pH to 7.0 with 5N NaOH. Sterilize by autoclaving.

4.8.3.

Grow bacteria in autoinduction medium: 25 g LB powder, 5 g yeast, add water to 1 L, Sterilize by autoclaving.

4.9.Buffers:

4.9.1.

Buffer A: 50 mM Tris-HCl, <strike>50 mM dextrose (glucose)</strike> and 1 mM EDTA, pH 7.9 (1000 mL)

4.9.2.

Buffer B: 10mM Tris-HCl, 50 mM <strike>KCl</strike> NaCl, 1 mM EDTA, <strike>1 mM PMSF</strike>, <strike>0.5% Tween-20</strike>, pH 7.9 (100 mL)

4.9.3.

Buffer A+B: 250 mL Buffer A with 250 mL Buffer B (500 mL), '''(25 mM KCl or NaCl''', pH 7.9)

4.9.4.

Buffer C: 20 mM <strike>HEPES</strike> Tris-HCl, '''50 mM''' <strike>KCl</strike> NaCl, 1 mM EDTA, <strike>0.5 mM PMSF</strike>, <strike>0.5% Tween-20,</strike> pH 7.9 (1500 mL)

4.9.5.

Buffer D: 20 mM <strike>HEPES</strike> Tris-HCl, 200 mM <strike>KCl, </strike> NaCl,1 mM EDTA, <strike>0.5 mM PMSF</strike>, <strike>0.5% Tween-20</strike>, pH 7.9 (800 mL)

4.9.6.

Buffer E: 20 mM <strike>HEPES</strike> Tris-HCl, 25 <strike>50 </strike>mM <strike>KCl</strike> NaCl, pH 8.3 (200 mL)

4.9.7.

Buffer F: 20 mM <strike>HEPES</strike> Tris-HCl, pH 8.3 (800 mL)

4.9.8.

Buffer G: 20 mM <strike>HEPES</strike> Tris-HCl, 550 mM <strike>KCl</strike> NaCl, pH 7.9 (200 mL)

5. Procedure

5.1. Grow and collect the Cell:

<strike>5.1.1. Plate the E.coli strain containing the Taq construct on LB agar containing 100ug/mL ampicilin.  Incubate the culture overnight at 37oC</strike>

<strike>5.1.2. Pick up one fat colony from overnight plate, inoculate into 2 mL LB containing 100 ug/mL ampiciling in the morning, then transfer to 200 mL LB containing ampicilin at evening, and grow overnight.</strike>

<strike>5.1.3. Transfer 15 mL overnight culture to 1L 2xYT containing 100 ug /mL ampicilin at morning (6L every time), and grow at 37oC with good aeration to OD595 0.6-0.8, then add IPTG to a final concentration of 1 mM and grow the culture overnight.</strike>

5.1.1. In the early morning (9AM), pick one colony (pET28b-Taq) from plate and grow bacteria in 4 ml LB/Kan

5.1.2. Make 8 L autoinduction medium, add 1 drop antiform to each flask.

5.1.3. In the afternoon (5 PM), add 0.5 ml bacteria to 1 L medium, grow at 37 oC O/N.

5.1.4. at about lunch time or after lunch harvest cells by centrifugation at 6000 rpm for<strike>10</strike> 5 min at 4oC using 500 mL centrifuge tube.

5.1.5. Resuspend the cell in Buffer A, and transfer to 50 mL conical tube, and collect the cell by centrifuge.

5.1.6. Weigh the cell pellet and record it. (70-80g is needed for purification)

5.2. Make the cell extraction (for 70-80 g of cell pellets):

5.2.1. After the pellets have thawed for 1 hr, resuspend them in the 500 mL Buffer A <strike>containing lysozyme (2 g lysozyme, Sigma)</strike> by pipeting at room temperature. Check the pH, and adjust to pH 7.9 if necessary.

5.2.2. Incubate for 15 min at room temperature with gentle shaking.

5.2.3. Add 500 mL Buffer B and <strike>2 mL 200mM PMSF</strike> to the resuspended cells and incubate the mixture in 75oC water bath for 30 to 60 min with frequent hand shacking.

5.2.4. Cell debris and denatured proteins are removed by centrifuging at 6000 rpm at 4oC for 15 min using 500 ml centrifuge tube, collect supernatant, spin supernatant at 12000 rpm for another 30 min, collect supernatant. Keep the supernatant at 4oC or -20oC if you want to stop here.

'''Optional'''

 Add neutralized 10% PEI  to supernatant to a final concentration of 0.05 to 0.8% and incubate the mixture  for 5 min at room temperature.

 Spin.

 Resuspend precipitate in buffer C (20 mM HEPES, pH 7.9,l mM EDTA, 0.5 InM PMSF, 0.5% Tween 20,0.5% Nonident-P40), 0.25 M KC1

5.2.5. Measure the total volume of cell extraction and record it. Save 0.5 mL aside for Taq QC assays later.

5.3. Preparation and Equilibration of the Bio-Rex 70 ion exchange column Note: If a Bio-Rex 70 column that has already been used and regenerated is to be used again for this SOP, skip step 5.3.11

^^^^^^^^^^^^^

5.3.1 Place approximately 500 g of Bio-Rex 70 resin in a container with a top and add 500 mL ddH2O and 5 mL 100%HCl. Allow the resin to equilibrate for 6-10 hr at room temperature, inverting several times about once an hour.

5.3.2 Measure the pH with pH paper and adjust the pH to 7.0 with 100% HCl, added dropwise while stirring at room temperature. Re-equilibrate for 12-20 hr at 4oC.

5.3.3 Stir the Bio-Rex 70 ion exchange resin with the pH meter probe and adjust the pH of to 7.0 with 100% HCl, added dropwise at room temperature.

5.3.4 Decant the water from the resin.

5.3.5 Add this 0.5M Tris_HCl, pH 7.9 to the resin. Allow the resin to equilibrate for 6-10 hr at room temperature, inverting several times about once an hour.

5.3.6 Stir the Bio-Rex 70 ion exchange resin with the pH meter probe and adjust the pH of to 7.0 with 100% HCl, added dropwise at room temperature. Re-equilibrate for 12-20 hr at 4oC.

5.3.7 Decant the buffer and add 500 mL of fresh 0.5M Tris-HCl, pH 7.9 buffer

5.3.8 Repeat this step until no pH change from pH 7.9 is seen when the fresh buffer is added. This may take several buffer changes.

5.3.9 Decant the excess buffer and pour the resin into a column with a diameter of 6 cm. Place the column at 4oC.

5.3.10 Allow the column resin to settle. Drain the buffer to a point approximately 3 cm above the resin bed. Note: Never allow the resin to dry out or allow the buffer to drain below the level of the resin.

5.3.11 Allow 30mM Tris-HCl pH 7.9 to pass through the column and check the pH of the effluent using pH paper. Continue the flow of 30mM Tris-HCl pH 7.9 until the pH of the effluent is 7.9-8.2.

5.3.12 Drain the buffer to a point approximately 3 cm above the resin bed.

^^^^^^^^^^^^^^

5.3.13 Apply Buffer A+B wash solution to wash the column and check the pH of the effluent using a pH meter or pH paper. When the pH of the effluent is 7.9, the resin is equilibrated and the column is ready to be loaded.

5.3.14 Store the column at 4oC for up to 7 days.

5.4. Running the Bio-Rex 70 column Note: Pre-chill all buffers used in this section to 4oC before running the column.

5.4.1. Allow access buffer A+B to pass through the column, leave enough buffer just to cover the top of the resin bed.

5.4.2. Load the cell extract dropwise to the top of the column without disturbing the resin bed. Adjust the liquid drop speed around 50 drops/min.

5.4.3. Drain the loading into the top of the bed and apply 500 mL Buffer A+B (25 mM KCl or NaCl, pH 7.9)to wash the column. Wash column with Buffer A+B until OD280 is <0.05 (or Bradford Ultra Assay)

5.4.4. Drain the Buffer A+B into top of bed and apply 1500 mL Buffer C (50 mM  KCl or NaCl, pH 7.9) to wash the column.'''Check the DNA contamination by agarose gel'''. The end of wash should not see genomic DNA on agarose gel.

5.4.5. Drain the Buffer C into the top of the bed and apply Buffer D (800mL) (200 mM <strike>KCl, </strike> NaCl) to elute the Taq.

5.4.6. Collect the first 320 mL of effluent in a 500 mL bottle. Then collect the effluents with 15 mL conical tubes. Check the OD260 and OD 280 of each collection. When the OD260/OD280>1 and the OD280>0.2, start to save the fractions until the OD280<0.2.

5.4.7. Combine the fractions with high purity and concentration together. Measure OD260, OD280 and the total volume and record them. Save 0.5 mL of it aside for Taq QC assays later.

5.4.8. Run PCR to check the activity of Taq in total combination.

5.5.'''Run the Q Sepharose Fast Flow Column'''

5.5.1.Allow access '''Buffer E '''to pass through the column, leave enough buffer just to cover the top of the resin bed.

5.5.2.Dilute the partially purified polymerase from the Bio-Rex 70 column purification four-fold with '''Buffer F''' ('''to a final concentration of 50 mM KCl'''). Load it into the top of the column without disturbing the resin bed.

5.5.3.Drain the loading into the top of the bed and apply 150 mL '''Buffer E''' to wash the column. Check the OD260 (it should be less than 0.1) and OD280 (it should be less than 0.1).

5.5.4.Drain the Buffer E into the top of the bad and apply '''Buffer G''' to elute the Taq.

5.5.5.Collect the effluents with 15mL conical tubes, Taq will come out after 40 mL. Check the OD260 and OD 280 of each fraction. Save all the fractions that have OD280>0.1.

5.5.6.The final OD280 should be between 1.8 and 2.2. Combine the fractions with OD260/OD280>0.8 and OD280>0.1. Measure final OD280 and OD260 and volume for combinations and record them. Save 0.5 mL of it aside for Taq QC assays later.
[[Category:Protocols]]

Taq purification - Revision history

Paul.xu at 13:23, 8 June 2016