Taq_production

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					                                Home Brew Taq
The volumes cited in this protocol relate to a 1L Taq culture. You can easily scale it up
depending on centrifugation capacity.

References

Engelke, D. R.; Krikos, A.; Bruck, M.E. & Ginsburg, D. (1990). Purification of Thermus
aquaticus DNA polymerase expressed in Escherichia coli. Analytical Biochemistry 191:
396-400.

Pluthero, F. G. (1993) Rapid purification of high activity Taq DNA polymerase. Nucleic
Acids Research. 21 (20): 4850-4851.

Ingredients
    E. coli strain (pRECTaqXLBlue) with pTaq plasmid (containing Taq gene
      expressed under control of the tac promoter).
    1L LB (700 mL, 30 mL, plus left over as back up and for spectrophotometer
      blank.)
    LB selective plates if you reckon you may like to isolate individual clones (use
      same concentration of Amp/Tet in plates as for the LB)

Item           Dissolve in      Stock conc.                  Supplier       Catalogue #
Ampicillin     H2O              100 mg/mL
Tetracycline 100% EtOH            5 mg/mL
IPTG           H2O               50 mg/mL
DTT           0.01M NaOAc pH 5.2 1 M
Complete tablets*                                            Roche     1 697 498
Dialysis tubing                                              GIBCO-BRL 15961-022

*Complete tablets are used instead of PMSF in the Pluthero protocol (1 tablet is sufficient
for one 50 mL of cell extract.) Highly toxic and unstable: make just prior to use.
Dissolve a tablet in 1 mL of buffer solution in a 1.5 mL eppie and vortex, then add back
to the main volume

Buffer A: 50 mM Tris-HCl (pH 7.9); 50 mM Dextrose; 1mM EDTA

Pre-Lysis Buffer: Buffer A plus 4 mg/mL lysozyme

Lysis Buffer: 10 mM Tris-HCl (pH7.9); 50 mM KCl; 1mM EDTA, Complete*; 0.5%
Tween 20; 0.5% Nonidet P40.

Storage Buffer: 50 mM Tris-HCl (pH7.9); 50 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.5
mM Complete*; 50% glycerol.
(optional – before starting this protocol, you may streak Taq stocks onto LB plates, grow
at 37 C overnight and use a single clone in this protocol)

Day 1. O/N culture

Prep 1L of LB, dispense into 30 mL and 700 mL and sterilise remaining volume in a
separate container (backup LB).

Just before leaving the lab… (eg. 5:00 pm)
Add 15 L of 100 mg/mL Ampicillin (50 g/mL) and 30 L of 5 mg/mL Tetracycline (5
g/mL), to 30 mL of LB contained in 250 mL Schott bottle.

Spike LB with TaqXL1 Blue clones using a sterile loop and incubate O/N at 37 C in a
shaking incubator (150 opm)

Day Two – Production of Taq

Dilute the O/N culture 1:100 ie. add 7 mL of the (30 mL) O/N culture to 700 mL of LB in
a 2L conical flask.

Glycerol prep the remainder of the culture and store at –80 C

Incubate for about 2 to 4 hours until the bacteria has reached log phase growth i.e,
OD600 = 0.2.

Induce expression of the Taq gene by adding 3.32 mL of a 50 mg/mL stock of IPTG
(1mM) to 700 mL culture

Grow until OD600 = 0.5 to 1.0. Check the OD periodically, ideally it should be 1.0 and is
expected to take 2 hours to grow that density – it always takes longer.

Turn water bath on to 75C (unless you are planning a short day) and chill centrifuge
tubes on ice.

Split the 700 mL culture between centrifuge tubes and spin the culture at 4K for 15
minutes. (keep cell culture in centrifuge tubes on ice.)

Wash the pellet ie. Decant supernatant and resuspend pellet in Buffer A (100 mL/L of
starting culture – i.e. 70 mLs)

Centrifuge as above, decant supernatant.

Freeze the pellet at –70 C for 20 mins. (incubation time may be extended if you wish to
centrifuge additional bacteria to increase the yield of Taq…. or to go home).

Thaw pellet, but do not let it sit for too long after this has happened.
Resuspend the pellet in 50 mL/L (of original culture, i.e.35 mLs total) of Pre-Lysis
Buffer. Keep buffer A on ice until it is needed.

After 15 minutes add an equal volume of Lysis Buffer. Incubate at 75 C for 1 hour.

Spin 15 000 rpm for 10 minutes at 4 C.

      From this step on: use super clean glassware and do not use any metal instruments
       or aluminium foil as metal ions can catalyse reactions.

Transfer and pool the supernatant to a pyrex beaker.

Recover Taq polymerase: SLOWLY Add 30g of Ammonium Sulfate/100mL of Room
Temperature lysate (should be about 70 mLs, therefore 21g – be exact in measurements).
A milky precipitate (Taq) should form.

Centrifuge the solution at 15 000 rpm for 10 min. and harvest the protein precipitate
using a plastic spatula. Take care as precipitate does not stick well to the side of the
tubes – looks like scum.

Resuspend the precipitate using a total volume of 20 mL of Buffer A per 100 mL of
original cleared lysate (should be about 14 mLs). Store on ice.

Dialysis

Cut 2 strips of 30 cm Dialysis tubing and prepare it thoroughly by washing it in MilliQ to
remove the “soapiness”.

Pipette Taq in buffer A into the 2 strips of Dialysis tubing with a double know tied at
both ends after excluding as much air as possible. Drop the bags into 1 L of storage
Buffer at 4 C. Leave O/N in a beaker containing a stirrer. Top beaker with glad wrap.

Day 3 – Dialysis

Prepare a fresh batch of Storage buffer, remove dialysis bags from beaker and change
solution. Place dialysis bags back into solution. Incubate at 4 C O/N again.

Day 4 – Aliquot and Storage of Taq at –80 C

At 4 C with Taq on ice…

Concentrate the Taq to one end of the bag and cut just beneath the knot using a sterile
blade. Transfer Taq to a 50 mL Falcon tube, add an equal volume of filter sterilised
storage buffer, mix gently and store. Minimise the contamination by the storage solution
on the outside of the bag.
Aliquot into sterile eppies and store at –80 C. First batch was 10 mLs. Based on the use
of 0.25 L of Taq/25 L PCR, you should have 40 000 units of Taq. I have successfully
used 0.15 L/ 30 L PCR. Therefore you may find that you can stretch it much further.

*A less refined version of this protocol is available, minus the dialysis

                Abbreviated Taq production method
*Follow regular protocol through the culturing and Taq expression steps.

Divide 700 mL culture among centrifuge tubes and spin at 4K for 15 mins.

Resuspend pellets in TE (10 mM Tris-Cl, 1mM EDTA) to 160 OD/mL
       ie. approximately 4 mLs

Sonicate on ice, avoid frothing (Lab B2: {1 x 40 secs; 2 x 20 secs} 50% Level 4
microtip)

Dilute with an equal volume of : (10 mM Tris-Cl pH8, 50 mM kCl, 0.5% NoniDet40,
0.5% Tween 20 1 mM EDTA)

Heat to 80C for 30 mins, cool on ice for 15 mins

Spin at 12K for 10 mins

Pipette of super and discard pellet

Add an equal volume of glycerol to the clarified extract

Store aliquots at –20C (long term storage at –80C)

Use 0.5 – 1 uL in 50 uL PCR reaction

Reference: Anal. Bioch. 191: 396 – 400 (1990)

This method will not remove all of the bacterial DNA and thus is not appropriate for use
in research that involves bacteria specific primers. It generally isn’t a problem for our
lab, but it might be for you if you are planning to give it to people who work with
bacteria. The dialysis method is much more thorough in purifying the taq, only it is
rather time consuming.

				
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