Berglund:Taq polymerase

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Purification of Taq polymerase

Before you start:
FYI: Taq polymerase when recombinantly expressed runs at about 95kDa on a SDS-Page gel.
Use the taq plasmid and transform into BL21 (DE3) pLyseS 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 (50µl of 34mg/ml) at 37°C
2. Make up your growth culture: 500ml LB + Amp (500µl of 50mg/ml) + chloramphenicol (250µl 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 1ml of 0.5M IPTG (fc=1mM) 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
21.125ml water

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 (the heating step denatures all of the other proteins)
8. Centrifuge at 12,000rpm 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)(Do not add until right before using!)
50% glycerol (750ml)
0.5% NP-40 (7.5ml of 100%)
0.5% Tween 20 (7.5ml of 100%)
703ml water

10. Dialyze overnight into the storage buffer. Use spectropor 15 kDA dialysis tubing (1cm width, ~30cm total) Volume will be reduced from 25ml to about 6ml. Double clip. Make sure your DTT is added fresh. We do not filter the storage buffer, but if you do you cannot filter the DTT, it binds to the filter. So add DTT after filtering.
11. Test the enzyme by running dilutions of the new Taq and comparing against a known sample of Taq. This will help you to determine how much to dilute your Taq into a working stock (this method generally makes enough Taq to dilute your 6ml into 300ml storage buffer total; we dilute 1ml aliquots at a time into 50ml working stocks). Don't forget to run the Taq with no template. Save 100ml of storage buffer for diluting. Taq polymerase in storage buffer is stored at -20°C

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 (do not have to add if doing Φ step as indicated above)
2.5mM MgCl2
500mM KCl (do not have to add if doing Φ step as indicated above)
0.5mM CaCl2

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 RQ1-DNase1 at 24µl (1µl/ml of protein 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 into the storage buffer. Volume will be reduced.