실험 Q&A > Etc.
Taq DNA polymerase 분리 및 정제 (Taq clone 분양)
한석균 (비회원)
250ml Centrifuge tubes (Nalgene) for cell harvest
45ml Centrifuge tubes (Nalgene Oakridge)
50ml Falcon tubes
4℃ high speed centrifuge machine
37℃ shaking incubator
Spectrophotometer for the cell growth and protein Qty.
2L flask with stopper
Water bath or hot plate (75℃)
30ml syringe for homemade column with cock and tubing
Column stand
250ml and 500ml beaker
Dialysis tubing (MWCO 6-8,000): cut 20cm length of tubing and soak into DI water and microwave to heat and remove glycerin (Spectra/Por 1 through 6 Standard RC membranes may require some extra preparation. While rinsing Spectra/Por 1 through 6 in water is typically sufficient to remove glycerin or preservative, Spectrum offers two membrane pre-treatment solution kits for the removal of the trace levels of heavy metals and sulfides introduced during manufacturing.)
Super broth for Taq clone [Trypton 32g (20%), Yeast extract 20g (1%), NaCl 5g (0.5%)] or Terrific Broth
Ampicillin 100mg/ml
Buffer A (50mM TrisHCl pH 7.9, 50mM dextrose, 1mM EDTA)
Lysis buffer or Buffer B (10mM TrisHCl pH 7.9, 50mM KCl, 1mM EDTA, 1mM PMSF*, 0.5% Tween 20, 0.5% Nonidet P40)
Dialysis buffer (20mM HEPES pH 7.9, 100mM KCl, 1mM EDTA, 1mM DTT*, 0.5mM PMSF*, 50% glycerol)
Storage buffer (50mM TrisHCl pH 7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mM PMSF*, 50% glycerol)
0.8M IPTG solution
1N NaOH
DEAE-cellulose (sigma D-3764, 100g): anion exchanger (capture anion molecules)
Ammonium sulfate (grind fine)
Lysozyme (store at 4℃)
Sterile DI water
*: add just before use
1) Super broth for Taq clone [Trypton 32g (20%), Yeast extract 20g (1%), NaCl 5g (0.5%)]
2) Autoclave and add 5ml of 1N NaOH, add ampicillin to 50-100ug/ml after cool down.
3) Make a seed culture (10ml) for inoculation and inoculate 10ml of seed culture to 1L super broth.
4) At Early log phase (3 hrs after inoculation), as OD600 0.6, add IPTG to 0.5mM as final concentration
5) After 16 hrs, harvest the cell (Do not over 16hours culture)
Extraction of Taq polymerase (500ml culture in 2L flask for making enough aeration)
1) Centrifuge 250ml of cultured broth with 6000~7000 rpm for 20mins at 4oC
2) Resuspend harvest cell with 20 ml of Buffer A for washing the cell and transfer the resuspended cell to new oakridge centrifuge tube (45ml size)
3) centrifuge again for cell harvest as same condition as above
4) discard supernatant and resuspend cell pellet with 20ml of buffer A with 400mg lysozyme
5) incubate the tube for 15 mins at room temperature (cell break)
6) add 20ml of Lysis buffer and incubate the tube at 75℃ water bath for 1 hour
7) centrifuge 15,000´g for 10 mins at 4℃
8) transfer clarified lysate to clean beaker (250ml volume, clean the beaker with DI water)
9) add (NH4)2SO4 (30g/ 100ml lysate) with stirring and then store at 4℃ overnight ((NH4)2SO4must be prepared as fine powder by grinding pestle in mortal.) while this incubation, wrap the top of the beaker with Saran wrap to protect the solution.
10) Transfer the solution into the 45ml centrifuge tube and spin the tube @ 15,000´g for 10 mins
11) creamy white color of protein layer will be formed and discard the liquid phase from the tube carefully.
12) Resuspend creamy white protein with10ml Buffer A (per 50ml original lysate) (If your lysate is 100ml, use 20ml of Buffer A)
13) Transfer the protein solution to the dialysis tubing (Spectra/Por membrane MWCO 6-8,000 flat width 32mm)
14) Dialyze the solution with 300-400ml of dialysis buffer for overnight (or until dialysis done)
15) Transfer the dialyzed protein solution to a fresh tube for column purification
For a new column, resuspend 10g of DEAE-cellulose in 100-200ml of DI water and microwave it for 2-3mins at high power. Mix DEAE resin solution and leave the solution to sink the DEAE cellulose at the bottom. Remove the top water layer and re-suspend DEAE with new DI water and heat it again with microwave as above. Do couple times of this washing step and wash DEAE-cellulose with 20mM Tris-Cl (pH 7.5~8.0) in final step.
Equilibrium buffer: 20mM Tris-HCl (pH 7.5~8.0)
1) 10g DEAE cellulose was added into 500ml dH2O
2) Gently agitate the beaker and settle down the DEAE
3) Decant supernatant (remove fine particle)
4) 2~3 times repeat in 3 step
5) Completely remove the water and fill the 20mM Tris-Cl in same volume
DEAE cellulose activation (recycle DEAE)
1) Add DI water to DEAE and degassing with sonicator for 1hour
2) Discard water phase and add 0.2N HCl for washing resin
3) Repeat washing with exchange to new 0.2N HCl (leave 2-3 hours for acidifying DEAE to remove DNA out)
4) Wash DEAE-cellulose with DI water.
5) Add 0.2N NaOH and try to neutralize the resin.
6) Repeat washing with exchanging 0.2N NaOH until get pH 7.0
7) Wash DEAE with DI water and wash it with starting buffer
8) Pack the column after degassing DEAE resin
Spectrophotometer & SDS-PAGE
1) Apply protein sample into the DEAE column and make flow rate as slow as possible
2) Flow 10mM, 50mM, 100mM NaCl / starting buffer and collect the effluent (3-5ml each effluent) (0.25ml/min)
3) Measure protein concentration with nanodrop (@ 275nm)
4) Check protein on 12% PAGE running
size marker (mid range) : 50~100ng
mix loading dye, then 10min boiling (sample)
® 120V, 2hr (50 kDa), 70 kDa 2.5 hrs, 90 kDa for 3 hrs
5) silver staining(or coomassie blue) ® 90 kDa band
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