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Taq DNA Polymerase Preparation

This document provides instructions for purifying Taq DNA polymerase from E. coli cells. The cells are grown, induced with IPTG, and lysed. The lysate is loaded onto an ion exchange column equilibrated with Buffer B. The column is washed and Taq polymerase is eluted with Buffer C. Eluted fractions containing Taq polymerase are pooled and dialyzed against Storage Buffer. Buffers and materials used include Terrific Broth media, Buffer A, B and C, and a Bio-Rex 70 ion exchange resin column.

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Ana Marina Santos
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67 views5 pages

Taq DNA Polymerase Preparation

This document provides instructions for purifying Taq DNA polymerase from E. coli cells. The cells are grown, induced with IPTG, and lysed. The lysate is loaded onto an ion exchange column equilibrated with Buffer B. The column is washed and Taq polymerase is eluted with Buffer C. Eluted fractions containing Taq polymerase are pooled and dialyzed against Storage Buffer. Buffers and materials used include Terrific Broth media, Buffer A, B and C, and a Bio-Rex 70 ion exchange resin column.

Uploaded by

Ana Marina Santos
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Sept 2010

Taq DNA Polymerase Preparation

1. Inoculate 1L TB medium (w/Ampicillin 100ug/ml) with 20ml of an overnight LB


culture (grown at 30°C) of Taq-bug (in DH5) and grew to
OD600= 0.4-0.5 at 37°C (200rpm)
2. Induce Taq expression by addition of of IPTG to a final conc. of 0.5mM (0.12g/l).
Continue growing for 16-20 hours
3. Harvest cells by 15min centrifugation at 3500rpm at 4°C
4. Resuspend cells in a total of 40ml with Buffer A
5. Add Lysozyme to a final conc of 4mg/ml and incubate at RT for 15min
6. Add 80ml Buffer B, mix and incubate for 1hr at 75°C in a shaking water bath
7. Pellet debris by centrifugation in a GSA rotor for 15min at 8000rpm at 4°C, discard
pellet

From now on work at 4°C

8. Add KCl to a final conc of 50mM


9. Load supernatant on the column with a flow rate of 2ml/min (could be done at RT
but buffers should be COLD)
10. Wash column with 2-3 volume ob Buffer B (or until basal level is reached)
11. Elute Taq DNA Polymerase with Buffer C. Collect 1.6ml fractions
12. Check peak fractions on a 10% SDS gel (size of Taq 85kDa), run gel o/n @ 30V
13. Pool fractions containing Taq and dialyse with Spectra/Por Float-A-Lyzer Dialyse
tubing several times (2x 1-2hrs; 1x o/n; 1x 1-2hrs) against Storage Buffer
 use to dyalize 30-40ml Taq Fractions 2 tubings only. Add first 10ml of Taq
Fractions, dialyse for 30min, fill up tuning again and so on.

A.Schauerte
Sept 2010

Column Preparation
Equilibrate 100g of Bio-Rex 70 ion exchange resin in Buffe B:
- put resin in a beaker, add 500ml Buffer B, stir for 30min
- adjust pH to 7.7-7.9, stir again and adjust pH until stable
- change buffer and repeat until pH is stable
- assemble Äkta column (Diameter 3cm; height 40cm), put the lower pistil 5cm
away
of the end
- poor resin in the column and let settle down by gravity flow
- wash column w/300-400ml Buffer B before loading the Taq lysate

Äkta Handling

Important: column should never run dry!!!!!

- connect column to Äkta, connect lower tube with UV detector and upper tube to Inj Valve 1
(with green tubing); check manual
- start up Computer
- switch on Äkta
- start program PrimeView

- on Äkta:
 select Manual Run
 set Methode on ml; Pressure max on 0.3bMPa; Buffer Valve Pos on 8
 use always tubing connected to Buffer Valve 8 to connect system w/ Buffer or
Taq Lysate
 set Injection Valve Pos on Waste (will not go over the column, but will wash the
system w/buffer), set Flow Rate to 20ml/min, connect w/ Buffer B; select Start Run
and wash 2-3min
 Press Pause, set Injection Valve Pos on Load (will go over the column) set Flow
Rate to 2ml/min, Press Continue wash column w/ Buffer B (around twice column
volume) until go got a stable signal

A.Schauerte
Sept 2010

 stop and start the system to start a new Chromatogramm (End-Start Run)
 load Taq Lysate w/ 2ml/min
 after Taq Lysate is loaded to column wash column w/ Buffer B until you reach again
the base line
 press Pause, connect system with Elution Buffer, set Injection Valve Pos on
Waste, press Continue and flush system w/ Elution Buffer
 press Pause, set Injection Valve Pos on Load, set Fraction Size on 1.6ml, press
Continue and start Fractioning
 stop Fractioning after you reached again the base line after the Elution peak

A.Schauerte
Sept 2010

Buffers and Material

Terrific Broth Media (TB): TB Salts:


12 g Bacto-tryptone 2.31 g KH2PO4
24 g yeast extract 12.54 g K2HPO4
4 ml glycerol ddH2O to 100 ml autoclave
ddH2O to 900 ml autoclave
 mix TB Media snd Salts before use!!

Buffer A
50mM Hepes pH 7.9
50mM Dextrose
1mM EDTA

Buffer B Buffer C
20mM Hepes pH 7.9 20mM Hepes pH 7.9
1mM EDTA 1mM EDTA
0.5% Tween-20 0.5% Tween-20
0.5% NP-40 0.5% NP-40
0.5mM fresh PMSF 0.5mM fresh PMSF
50mM KCl 200mM KCl

Storage Buffer
50% Glycerole
1% Triton X-100
50mM Tris-Cl pH 8.0
100mM NaCl
0.1mM EDTA
5mM DTT

A.Schauerte
Sept 2010

Resin: Bio-Rex 70 Weakly Acidic Analytical Grade Cation Exchange Resin


200-400mesh sodium form (BioRad 143-5852)

Dialysis Membran: Spectra/Por Float-A-Lyzer G2, mulecularporous membran tubing


(MWCO 20000; Diameter 10mm; Volume 10ml, G235069)

Äkta Column: Diameter 3cm; height 40cm

10% SDS Gel:

A.Schauerte

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