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PCR Primer Design

HOW TO DESIGN Primers for PCR

Guidelines for primer selection and software design:

  • PCR primers are designed as 15-30 oligo nucleotides in length. Longer primers usually allow higher specificity or allow the addition of restriction enzyme sites to the primer end for cloning.
  • Primer oligonucleotide GC content should be in the range of 40-60%. G and C nucleotides should be uniformly distributed as clusters of G's or C's cause non-specific priming may occur and high GC content impairs the PCR efficiency.
  • Primers should not be self-complementary or complementary to the other primer in the reaction mixture. Primers that complement with each other compete with the template for the use of primer and reagent. These are called primer dimers and hairpin formation when a primer self-complements.
  • The melting temperature Tm of the primers should not have a difference more than 5°C.

PCR Primer Design Software Online

Recommended Primer Design Software:

WebPrimer Picks the best pair of primers from many conditions which can be set manually.

Primer3 Primer3 primer design software picks primers from a DNA sequence. Picks primers from many conditions which can be set manually.

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loading dye Has anybody ever come across a loading dye that ran at a different molecular size on an agarose gel than expected? Why is this happening?
Cresol Red/Sucrose Hi. Does anyone have experience using cresol red/sucrose as loading dye added to the reaction BEFORE PCR? Does cresol red/sucrose have to have a...
Amplification won't work! I'm trying to amplify a ~200bp segment from a ~4kb plasmid. A postdoc in my lab previously amplified the segment and gave me a stock of gel-extracted...
Any ideas why my PCR amplification won't work? I'm trying to amplify a ~200bp segment from a ~4kb plasmid. A postdoc in my lab previously amplified the segment and gave me a stock of gel-extracted...
Heated lid on RFLP: is it ok? I'm doing three different RFLPs with three different restriction enzymes using a single amplicon, each RFLP in different tubes of course as each...
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