Reagent Concentrations: Primers: 0.2 - 1.0 uM 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. |
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