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| <h3><font style="color:#F8B603;">Purification of Taq Polymerase</font></h3>
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| From Chory lab, taken from Pluthero, F.G. (1993) NAR 21: 4850-4851
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| Note: Rinse out all glassware with DI H<sub>2</sub>O (to get rid of detergent residue) before use in this protocol.
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| #Inoculate an overnight culture of the E. coli strain INVJαF’ containing the pTq plasmid [Engelke, D.R. et al. (1990) Anal. Biochem. 191: 396-400] in LB. (In our lab this is pTaq, glycerol stock IJ3; carb resistance.) Use 100ul of this overnight culture to inoculate a 1L culture.
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| #Grow 1L culture 11 hours (OD approx. 0.8) and add IPTG to final concentration of 124mg/L. Induce at 37° for 12 hours with shaking (longer induction times lead to degradation of the enzyme.)
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| #Harvest cells by centrifugation and wash in 100ml buffer A (see below). Recover cells and resuspend in 50ml buffer A plus 4mg/ml lysozyme. Incubate 15 minutes at room temperature.
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| #Add an equal volume (50ml) of lysis buffer (see below) and incubate at 75° for 1 hour. Clear lysate by centrifugation at 10,000 rpm in SS34 for 15 minutes at 4° (do NOT use blue-cap tubes – they will crack at such high speeds!).
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| #To cleared lysate, slowly (over the course of ~30 minutes) add 30g ammonium sulfate ((NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>) per 100ml with stirring (enzymes denature at air/liquid boundaries ==> avoid bubbles! We suggest using the smallest stir bar available on low setting) at room temperature. Let stir an additional half hour. Centrifuge this solution as in step 4 and harvest protein ppt (some will be in a pellet, but much of it will be on the surface and stuck to the walls.)
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| #Resuspend protein in 20ml buffer A per 100ml cleared lysate and dialyze 2x12 hours (we use slide-a-lyzers overnight) against storage buffer (see below).
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| #After dialysis, dilute the resulting protein 1:1 with storage buffer. Run PCR using a dilution series of this new taq (dilute with storage buffer). Determine appropriate dilution for use, dilute if necessary, aliquot and store at -80°.
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| <b>Buffer A</b>
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| 50 mM Tris-HCl pH 7.9<br>
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| 50 mM dextrose<br>
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| 1mM EDTA<br>
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| <b>Lysis Buffer</b>
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| 10 mM Tris-HCl pH 7.9<br>
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| 50 mM KCl<br>
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| 1mM EDTA<br>
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| 1mM PMSF*<br>
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| 0.5% Tween-20<br>
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| 0.5% Nonidet P40<br>
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| <b>Storage Buffer</b>
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| 50 mM TrisHCl pH7.9<br>
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| 50 mM KCl<br>
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| 0.1 mM EDTA<br>
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| 1 mM DTT<br>
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| 0.5 mM PMSF*<br>
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| 50% glycerol<br>
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| <nowiki>*</nowiki> Or Pefabloc at 1mM working conc. 100mg/ml = 0.4M Pefabloc; 25mg/ml = 0.1M Pefablock<br>
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| Sterilization: Filter sterilize Buffer A (do not autoclave). Lysis Buffer does not need to be autoclaved if used right away (within 24 hours). Do NOT add PMSF/Pefabloc or DTT until AFTER autoclaving. Chill buffers before use.
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