Taq purification

Taq DNA polymerase purification

This protocol yields a Taq polymerase that is pure enough for most purposes that do not involve amplification of E. coli DNA or some DNA that is common to pBR322 derived plasmids as there will be contamination with E. coli chromosomal DNA and the expression plasmid. This can be resolved by always including a negative control without template.

1. Streak the Taq E. coli strain on amp plate (box: Björn#2 position #9)
2. Pick a single colony and inoculate 2 ml Superbroth (SB)
3. Grow O/N
4. Inoculate 500 ml SB with 1ml of previous culture
5. Grow to OD640 = 0.4
6. Add 1ml 1M IPTG (2 mM final conc)
7. Grow another 16-20 hours
8. Harvest cells and wash once with 100 ml Buffer A. (8000 rpm for 10 min 4C or fast and long enough to collect the cells efficiently)
9. Resuspend cells in 20 ml buffer A + 4 mg/ml hen egg lysozyme added as a powder
10. Incubate at RT 15 min
11. Add 20 ml buffer B (add PMSF just before using the buffer)
12. Incubate at 75C for 60 min
13. Spin down debris at 25000 rpm 15 min 4C
14. Transfer supernatant to a clean tub, be careful not to transfer solid material
15. Mix lysate with an equal volume of storage buffer containing 50% glycerol.
16. Mix the diluted lysate with an equal volume of storage buffer containing 75% glycerol.
17. This brings the final glycerol concentration to 50% and diluting the lysate 4 times.
18. Note! Slow addition of both storage buffers (with mixing) is supposed to result in more units of Taq pol
19. Dispense the mixture in Eppendorf tubes and store at -20 C.

Superbroth (SB) (1L)

• 32 g tryptone
• 20 g yeast extract
• 5 g NaCl
• 1.25 ml 4M NaOH

Buffer A

• 50 mM Tris-HCl, pH 8
• 50 mM dextrose
• 1 mM EDTA

Buffer B

• 10 mM Tris-HCl, pH 8
• 50 mM KCl, 1 mM EDTA
• 0.5% Tween 20
• 0.5% Nonidet P40 (if you do not have Nonidet P40, take twice of the Tween)
• 1 mM PMSF

Storage buffer

• 50 mM Tris-HCl, pH 8
• 100 mM NaCl
• 0.1 mM EDTA
• 0.5 mM DTT
• 1% Triton X-100
• 50% glycerol OR 75% glycerol

Stock solutions:

• 1M Tris-HCl pH 8
• 500mM EDTA pH 8
• Glycerol 50% v/v
• Glycerol 75% v/v
• 100mM PMSF in DMSO
• Tween 20
• Nonidet P40 (if you do not have Nonidet P40, take twice of the Tween)

References and resources:

http://mama.indstate.edu/pfaffle/ptaq