This is a protocol from Engelke et.al.(1990)which was modified by Dr.Baron.The enzyme has been used for RT-PCR,genotyping and cloning.

Solutions

1000X IPTG

0.5M IPTG 1.19g IPTG

up to 10 ml wih sterile Q

filter and store at -20℃

Buffer A

50 mM Tris 7.9 25 ml 1M Tris pH 8.5*

50 mM dextrose 4.5 g dextrose

1 mM EDTA 1 ml 0.5M EDTA

up to 500 ml with Q

*Check pH and bring to 7.9 as the dextrose will reduce the pH.

Store at room temperature.For some steps add Lysozyme to a

final concentration of 4 mg/ml.

Buffer B

10 mM Tris 7.9 5 ml Tris pH 7.9

50 mM KCl1.86 g KCl

1 mM EDTA 1 ml 0.5M EDTA

0.5% Tween 20 5 ml 50% Tween 20

0.5% NP-40 5 ml 50% NP-40

up to 500 ml with Q

Store at room temperature and add PMSF to a final concentration of 1 mM just prior to use.

Buffer C

50 mM Tris 8.0 50 ml 1M Tris pH 8.0

50 mM KCl3.72 g KCl

1 mM EDTA 2 ml 500 mM EDTA pH 8.0

50% glycerol 500 ml glycerol

0.5% Tween 20 5 ml 50% Tween 20

0.5% NP-40 5 ml 50% NP-40

1 mM DTT 2 ml 0.5M DTT

1 mM PMSF 10 ml 100 mM PMSF

up to 1 liter with Q

Buffer D

50 mM HEPES 7.9 50 ml 1M HEPES pH 7.9

50 mM KCl3.72 g KCl

5% glycerol 100 ml 50% glycerol

1 mM EDTA 2 ml 0.5M EDTA 8.0

0.5% Tween 20 10 ml 50% Tween 20

0.5% NP-40 10 ml 50% NP-40

1 mM DTT 2 ml 0.5M DTT

1 mM PMSF 10 ml 100 mM PMSF

up to 1 liter with Q

Procedure

? Streak out the E.coli strain carrying the Taq polymerase gene on an LB amp plate and innoculate a 5 ml overnight culture with a single colony.

? Innoculate a 1 liter culture of LB-amp with the 5 ml of overnight culture and grow to an A600 of 0.2.Add IPTG to a final concentration of 5 mM and culture overnight.

? Spin down the culture and resuspend the pellet in 200 ml of Buffer A (ice cold).

? Spin down the E.coli as before and resuspend in 50 ml Buffer A containing 4 mg/ml lysozyme.Incubate at room temperature for 15 minutes.

? Add 50 ml Buffer B and incubate at 75℃ for 1 hour.

? Chill on ice and spin in the GSA rotor at 8,000 rpm for 15 minutes.

? Transfer the supernatant to a beaker on ice.Measure the volume and add pulverized ammonium sulfate slowly to a final concentration of of 0.164 g/ml (30% saturation).Continue to stir for 30 minutes.

? Spin down the precipitate by spinning in the SA-600 rotor at 13,000 rpm for 30 minutes at 4℃.

? Transfer the supernatant to a clean beaker on ice,measure the volume and add ammonium sulfate to a final concentration of 0.181 g/ml (60% saturation).Stir as before for 30 minutes on ice.

? Spin down the precipitate in the SA-600 rotor at 13,000 rpm for 30 minutes at 4℃ and resuspend in 10 ml Buffer D.

? Equilibrate a DEAE-sephacel column (1.5 cm diameter,5 diameter height,9 ml bed volume)in Buffer D.

? Load protein and wash with 3-5 column volumes of buffer D.

? Elute in 20 ml buffer D containing 0.5M KCl and collect the first six 3 ml fractions.

? Dialyze fractions 2,3,4 against 1 liter of Buffer C containing 50% glycerol for 4-8 hours and repeat.This should be done on ice in the cold room.

? Freeze in small aliquotes and store at -80℃.