Standard PCR Protocol

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 - 94oC for 30 - 60 sec (denature) 　　37 - 72oC for 30 - 60 sec (anneal) 　　72oC for 30 - 60 sec (elongate) (60 sec per kb target sequence length) 　　25 - 35 cycles only (otherwise enzyme decay causes artifacts) 　　72oC for 5 min at end to allow complete elongation of all product DNA