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How can I improve the PCR output of a low concentrated DNA?
Posted November 24, 2020
Chemical ReagentsDNA and RNAPhysiological Probes
Answer

If you are experiencing low yield of PCR product, then try the following to improve output:

  • If the concentration of your DNA sample is too low, then use ethanol precipitation to concentrate your DNA sample
  • Increase the number of PCR cycles to 45
  • Optimize your annealing temperature
  • Optimize your primer concentration, the concentration of each primer should range between 0.1 to 0.5 µM, but for most PCR applications 0.2 µM is sufficient
  • Optimize your dNTPs concentration, each dNTPs should have a concentration of 200 µM
  • Check primer design
Additional resources
Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategiesROX Reference Dye *50X fluorescence reference solution for PCR reactions*ROX Reference Dye for Real-Time PCRReverse Transcription PCR (RT-PCR)

What is next-generation sequencing (NGS)?What is the acceptable PCR primer efficiency value?What is stringency in hybridization?What is end-point RT-PCR?6-ROX is predominately used as a reference dye for performing PCR detections, however, 6-ROX is very unstable compared to other rhodamine dyes. What is a better dye replacement?