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. DMSO apparently allows better denaturation of longer target sequences (>1kb) and more product.ĭO NOT USE SAME PIPETTE FOR DISPENSING NUCLEIC ACIDS AS YOU USE FOR DISPENSING REAGENTS Use of detergents is recommended only for Taq from Promega (up to 0.1% v/v, Triton X-100 or Tween-20). USE PLUGGED PIPETTE TIPS: prevents aerosol contamination of pipettes. NOTE: latest wisdom has it one can use VASELINE - this also allows "HOT START" PCR. OVERLAY REACTIONS WITH 50UL OF HIGH-QUALITY LIQUID PARAFFIN OR MINERAL OIL to ensure no evaporation occurs: this changes reactant concentrations. 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. Promega now supply 25mM MgCl2, to allow user-specified for reaction optimisation with different combinations of primers and targets. This should contain: minimum of 1.5mM Mg2+, usually some detergent, perhaps some gelatin or BSA. Target DNA: 1 ng - 1 ug (NB: higher concn for total genomic DNA lower for plasmid / purified DNA / virus DNA target)īuffer: use proprietary or home-made 10x rxn mix eg: Cetus, Promega. Taq polymerase: 0.5 - 1.0 Units/50ul rxn.Dimethyl sulphoxide (DMSO): 0 - 10% (v/v).Labelling PCR Products with Digoxigenin.
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