Griffitts:PCR primers

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Contents

Primer Design

General

  • Primers should generally be 20–22 nt long with 10–12 Gs and Cs
  • Remember to use the reverse compliment for your reverse primer

Sequencing

  • Design one forward primer for every ~500 bp
  • You won't get good sequence where your primer sits, so design your first primer to be a little upstream from your sequence
  • You only need one reverse primer

Cloning

  • Check for restriction sites within your target before adding them to your primers
  • Add a "CGC" to the ends of your primers

Gene Deletion

  • Check for restriction sites within your target before adding them to your primers
  • Add a "CGC" to the ends of your primers
  • Clone ~500 bp upstream and downstream of the gene you're intending to delete and include ~20 bp of the target gene
  • For your internal primers, give them 10-12 bp homology with each other for Overlap-extension PCR

Primer Hydration

  • Find the nmoles on the tube (e.g. 24.65 nmoles)
  • Multiply by ten (e.g. 24.65 × 10 = 246.5)
  • Take that number and add that many μL of TE buffer (e.g. add 240 to 250 μL)
  • This will give you 100X primers
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