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PCR Calculator

Calculate master mix, cycling conditions, and optimize PCR reactions

Template & Reaction Setup

Primer & Cycling Conditions

Used for annealing temperature optimization

Quick Tm Calculator

Short primers (<20 bp): Tm = 2x(A+T) + 4x(G+C)
Long primers: Tm = 64.9 + 41x(G+C-16.4)/length
Annealing temp = Tm - 5 C (typical)

Troubleshooting Guide

No Product / Weak Bands

  • - Increase template amount (try 10-500 ng)
  • - Lower annealing temperature by 2-5 C
  • - Extend annealing/extension times
  • - Check primer design and specificity
  • - Add PCR enhancers (DMSO, betaine)
  • - Increase cycle number (up to 40)

Non-specific Products

  • - Increase annealing temperature by 2-5 C
  • - Decrease template amount
  • - Use hot-start polymerase
  • - Optimize primer concentrations
  • - Reduce cycle number
  • - Use touchdown PCR protocol

General Tips

  • - Always include positive and negative controls
  • - Template quality is critical - check A260/A280 ratio
  • - Store master mix on ice and add template last
  • - Use PCR-grade water and clean technique
  • - Consider primer dimers if using low template amounts
  • - For GC-rich templates, add DMSO (2-10%) or use specialized polymerases