Berglund:Taq polymerase
Purification of LabTaq1
Before you start:
Use the taq plasmid and transform into BL21 (DE3) pLyse S cells
Treat your LB+ Amp plates with chloramphenicol (overlay the top with 10µl 34mg/ml Chloramphenicol diluted into 100ml LB + Amp broth, let dry)
Use glass beads to plate the transformed cells
1. Pick one colony and set up an overnight culture of the plasmid in 50ml LB + Amp (100µl of 50mg/ml) + chloramphenicol (200µl of 34mg/ml) at 37°C
2. Make up your growth culture: 500ml LB + Amp (500µl of 50mg/ml) + chloramphenicol (200µl of 34mg/ml)
3. Innoculate 500ml of LB with 3ml of overnight culture and grow at 37°C to an O.D. 600= 0.4
4. Add 250µl of 0.5M IPTG and grow for 16 hours at 37°C
5. Harvest cells by centrifugation at 6000 rpm for 15 minutes
Buffer A
50mM Tris-HCl, pH 7.9 (1.25ml of 1M)
50mM glucose (2.5ml of 0.5M)
1mM EDTA (0.125ml of 0.2M)
4mg/ml (100mg)lysozyme
water (21.125ml)
fv=25ml
6. Resuspend the pellets in 12ml of Buffer A and incubate at room temperature for 15 minutes with rocking
Buffer B
10mM Tris-HCl, pH 7.9 (0.25ml of 1M)
50mM KCl (1.25ml of 1M)
1mM EDTA (0.125ml of 0.2M)
0.5% Tween 20 (0.125ml of 100%)
0.5% NP-40 (IGEPAL)(0.125ml of 100%)
23.125ml water
fv= 25ml
7. Add 12ml of Buffer B and incubate 60 minutes at 75°C
8. Centrifuge at 12,000rcf for 10 minutes
9. Collect the supernate, discard the pellet. Save some supernate to do PCR with to make sure that dialyzing the Taq into the storage buffer doesn't reduce it's activity
Storage buffer (store in fridge before use)
20mM Tris-HCl, pH 8 (30ml of 1M)
100mM KCl (11.18g)
0.1mM EDTApH 8 (0.3ml of 0.5M)
1mM DTT (0.75ml of 2M)
50% glycerol (750ml)
0.5% NP-40 (7.5ml of 100%)
0.5% Tween 20 (7.5ml of 100%)
703ml water
fv=1500ml
10. Dialyze overnight back into the storage buffer. Volume will be reduced from 25ml to about 6ml.
11. Test the enzyme by running several known PCR products and compare the DNase treated enzyme versus the untreated with and without template.
Optional DNase step (do this before dialyzing into storage buffer if you prefer)
1. Take the enzyme and dialyze overnight carefully into DNase digestion buffer in a cold room with active stirring:
10x DNase digestion buffer
200mM Tris pH 8.4
20mM MgCl2
500mM KCl
Note: Leave lots of room in the dialysis bag and double clip it. The bag will expand a lot due to osmotic pressure.
2. Empty the dialysis bag into a 50ml conical tube
3. Add RNase free DNase1 at roughly 100units per ml (you will want to base this on the original volume before dialysis)
4. Incubate at 30°C for 10 minutes
5. Incubate at room temperature for 30 minutes
6. Heat kill the DNase at 70°C for 30 minutes
7. Dialyze overnight back into the storage buffer. Volume will be reduced.
