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 Mg 2+ 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 - 97℃ for 3 - 5 min. If you denature at 97℃, 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 - 75℃). Stringent initial conditions mean less non-specific product, especially when amplifying from eukaryotic genomic DNA.
Initial elongation temperature: 72℃ for 3 - 5 min. This allows complete elongation of product on rare templates.
