Recommended Reagent Concentrations:

• Primers: 0.2 - 1.0 uM

• Nucleotides: 50 - 200 uM EACH dNTP

• Dimethyl sulphoxide (DMSO): 0 - 10% (v/v)

• Taq polymerase: 0.5 - 1.0 Units/50ul rxn

Target DNA: 1 ng - 1 ug (NB: higher concn for total genomic DNA; lower for plasmid / purified DNA / virus DNA target)

Buffer: use proprietary or home-made 10x rxn mix; eg: Cetus, Promega. This should contain: minimum of 1.5mM Mg2+, usually some detergent, perhaps some gelatin or BSA. Promega now supply 25mM MgCl2, to allow user-specified [Mg2+] for reaction optimisation with different combinations of primers and targets.

MAKE POOLED MASTER MIX OF REAGENTS IN ABSENCE OF DNA using DNA-free pipette, then dispense to individual tubes (using DNA-free pipette), and add DNA to individual reactions USING PLUGGED TIPS.

OVERLAY REACTIONS WITH 50UL OF HIGH-QUALITY LIQUID PARAFFIN OR MINERAL OIL to ensure no evaporation occurs: this changes reactant concentrations. NOTE: latest wisdom has it one can use VASELINE - this also allows "HOT START" PCR.

NOTE:

USE PLUGGED PIPETTE TIPS: prevents aerosol contamination of pipettes.

Use of detergents is recommended only for Taq from Promega (up to 0.1% v/v, Triton X-100 or Tween-20). DMSO apparently allows better denaturation of longer target sequences (>1kb) and more product.

DO NOT USE SAME PIPETTE FOR DISPENSING NUCLEIC ACIDS AS YOU USE FOR DISPENSING REAGENTS

Remember sample volume should not exceed 1/10th reaction volume, and sample DNA/NTP/primer concentrations should not be too high as otherwise all available Mg2+ is chelated out of solution and enzyme reactivity is adversely affected. Any increase in dNTPs over 200uM means [Mg2+] should be re-optimised.

AVOID USING EDTA-CONTAINING BUFFERS AS EDTA CHELATES Mg²⁺

Low primer, target, Taq, and nucleotide concentrations are to be favoured as these generally ensure cleaner product and lower background, perhaps at the cost of detection sensitivity.

Recommended Reaction Conditions:

Initial Conditions:

Initial denaturation at start: 92 - 97oC for 3 - 5 min. If you denature at 97oC, denature sample only; add rest of mix after reaction colls to annealing temperature (prevents premature denaturation of enzyme).

Initial annealing temperature: as high as feasible for 3 min (eg: 50 - 75oC). Stringent initial conditions mean less non-specific product, especially when amplifying from eukaryotic genomic DNA.

Initial elongation temperature: 72oC for 3 - 5 min. This allows complete elongation of product on rare templates.

Temperature Cycling:

• 92 - 94^oC for 30 - 60 sec (denature)

• 37 - 72^oC for 30 - 60 sec (anneal)

• 72^oC for 30 - 60 sec (elongate) (60 sec per kb target sequence length)

• 25 - 35 cycles only (otherwise enzyme decay causes artifacts)

• 72^oC for 5 min at end to allow complete elongation of all product DNA

NOTE:

"Quickie" PCR is quite feasible: eg, [94^oC 30 sec / 45^oC 30 sec / 72^oC 30 sec] x 30, for short products (200 - 500 bp).

YOU CAN USE GLYCEROL IN THERMAL CYCLER REACTION TUBE HOLES TO ENSURE GOOD THERMAL CONTACTS

DON'T RUN TOO MANY CYCLES: if you don't see a band with 30 cycles you probably won't after 40; rather take an aliquot from the reaction mix and re-PCR with fresh reagents.  See here.

Labelling PCR Products with Digoxigenin

PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991; Nucleic Acids Res 19: 2790). This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions. It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.

Make a DIG-dNTP mix for PCR as follows:

DIG NUCLEOTIDE MIX CONCENTRATIONS

• Dig-11-dUTP 350 uM

• dTTP 650 uM

• dATP 1 mM

• dCTP 1 mM

• dGTP 1 mM

For each 50 ul of probe synthesized, a 1/10 dilution is made of the DIG-nucleotide mix when added to the other reagents as described above. The products may be analyzed by agarose gel electrophoresis - NOTE: PRODUCTS ARE LARGER THAN NON-SUBSTITUTED PRODUCT - and detected directly on blots immunologically. Probes can be used as 5-10 ul aliquots directly from PCR product mixes, mixed with hybridisation mix and denatured. Probes can be re-used up to 10 times, stored frozen in between experiments and boiled to denature.

Cleaning PCR Products

• Getting rid of mineral oil: simply add 50ul of chloroform to the reaction vial, vortex and centrifuge briefly, and remove the "hanging droplet" of AQUEOUS solution with a micopipette.

• Getting rid of wax or Vaseline: simply "spear" wax gem and remove; do as for oil or bottom-puncture tube and blow out aqueous drop for Vaseline.

• Cleaning-up DNA: 3 options

• a protocol which gives DNA that is clean enough for sequencing is the following: increase volume to 100ul with water, add 10M ammonium acetate soln. to 0.2M final concentration (1/5th volume), add equal volume of isopropanol (propan-2-ol), leave on bench 5 min, centrifuge 20 min at 15 000 rpm, remove liquid using pipette, resuspend in 100ul water or TE, repeat precipitation.

• Simply do a phenol-CHCl3 extraction (add 20ul phenol to aqueous phase, vortex, add 50ul CHCl3, vortex, centrifuge, remove UPPER aqueous phase, repeat CHCl3 extraction).

• Make aqueous phase up to 400ul, and spin through Millipore Ultrafree-MC NMWL 30 000 cartridges (at 6000 rpm in microcentrifuge), wash through with 2x400ul water, collect +/-20ul sample: this is pure enough for sequencing.

NOTE:

Product is clean enough for restriction digest immediately after reaction; however, phenol-chloroform clean-up is recommended as a minimum.

Sequencing PCR Products:

This is best done using ssDNA generated by asymmetric PCR, and the "limiting" primer for sequencing. However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. (1990) (NAR 18: 1309). CLEAN DNA is resuspended in sequencing buffer containing 0.5% Nonidet P-40 and 0.5% Tween-20 and sequencing primer, denatured by heating to 95oC for 5 min, snap-cooled on wet ice, and sequenced by the "close-to-primer" protocol (eg: dilute extension mixes).