Smolke:Protocols/Screening

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Contents

Josh's Protocol

Sample Preparation

  • Aliquot 50 uL sterile water into sterile PCR tubes, one tube per colony.
  • Using a P20, scape off a colony and resuspend it in the water.
  • Use 1 uL of this cell suspension as your PCR template
    • Tubes can be stored at 4C for several days. Use 5 uL of cell suspension to inoculate a 5 mL overnight culture.

Reaction Mixture

  • Per colony:
    • 1 uL cell suspension
    • 0.2 uL dNTPs
    • 0.2 uL FWD and REV primers
    • 1 uL 10x Taq Buffer (with MgCl2)
    • 0.2 uL 1:10 Taq
    • 7.2 uL water
  • Note: Make a master mix of everything except the template. Aliquot 9 uL into a separate set of PCR tubes. Then transfer 1 uL of cell suspension to each tube. You can use a multichannel pipet to transfer up to eight templates at once.
  • Run PCR as normal, with the first step changed to a 7 minute incubation at 95C (5 minutes to lyse + 2 minutes to denature DNA).


Yvonne's Protocol

Procedure

  • Warm up necessary number of LB (+ appropriate antibiotics) plates for restreak
  • Make master mix; include 1 or 2 extra volumes to account for errors from repetitive pipetting. Remember to account of positive and negative control samples. For each sample:
    • 19.5 ul water
    • 2.5 ul 10x Taq buffer
    • 1.25 ul 50 mM MgCl2
    • 0.5 ul Fwd primer
    • 0.5 ul Rev primer
    • 0.5 ul dNTP
  • Line up necessary number of PCR tubes
  • Take out pre-warmed LB plate and label with colony numbers
  • Add 0.25 ul 1:10 Taq DNA polymerase for each sample to master mix; pipette or invert tube to ensure thorough mixing
  • Aliqout 25 ul of master mix to each tube
  • Pick up each colony with a toothpick, dip into PCR tube (containing master mix) and swirl, then restreak on LB plate
  • Run PCR as normal, except the first step is changed to 95C for 5 min for cell lysis
    • Note: The second step is 94C for 2 min, and this step is repeated each cycle while the first lysis step is not
  • Put restreaked plate back in incubator; restreaked colonies should be ready for inoculating liquid cultures in 4 hours
  • Load 15 ul of each PCR product on agarose gel for analysis

Notes

  • Ideally, use primer sets that would generate a band for both positive and negative colonies but with different sizes.
  • Should always include a negative control using the parent vector, no DNA template, or another appropriate choice. This would let you know if you have primer dimer or other nonspecific amplification.
  • Should include a positive control if possible. This is especially important if your chosen primers would only generate a band for positive but not negative colonies.
  • I use toothpicks whenever possible. They are much cheaper and more environmentally friendly (I believe) than pipet tips.
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