Berglund:LabTaq1

Purification of LabTaq1

1. Restreak from the freezer stock onto a Lb-4xAmp plate and grow overnight at 37°C 2. Pick one colony and set up an overnight culture of the plasmid in 50ml LB + Amp at 37°C 3. Innoculate 500ml of LB+Amp with 1ml of overnight culture and grow at 37°C to an O.D. 600= 0.4 4. Add IPTG to a final concentration of 1mM and grow for 16 hours at 37°C 5. Harvest cells by centrifugation at 6000 rpm for 15 minutes 6. Resuspend the pellets in 12ml of Buffer A and incubate at room temperature for 15 minutes

Buffer A 50mM Tris-HCl, pH 7.9 50mM glucose 1mM EDTA 4mg/ml lysozyme

7. Add 12ml of Buffer B and incubate 60 minutes at 75°C

Buffer B 10mM Tris-HCl, pH 7.9 50mM KCl 1mM EDTA 0.5% Tween 20 0.5% NP-40 (IGEPAL)

8. Centrifuge at 12,000rcf for 10 minutes 9. Collect the supernate, discard the pellet

DNase step

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.

Storage buffer 20mM Tris-HCl, pH 8 100mM KCl 0.1mM EDTA 1mM DTT 50% glycerol 0.5% NP-40 0.5% Tween 20

8. Test the enzyme by running several known PCR products and compare the DNase treated enzyme versus the untreated with and without template.