Surveyor Assay for Genomic DNA Mutations


Materials

  • QuickExtract Reagent
  • Platinum PCR SuperMix High Fidelity (Life Technologies cat# 12532-016)
  • Amplification Primers (2 uM stock solution with both primers combined)
  • Surveyor Assay Kit (IDT cat# 706021)

Protocol

DNA Extraction

  • Aspirate media and add 50 uL QuickExtract Reagent to a 24 well of cells. No need to wash cells.
  • Place in PCR tube and digest using quick-extract PCR program:
    • 65C 10 minutes
    • 68C 15 minutes
    • 98C 10 minutes
  • Store at -20

Amplification of Target Region

  • Using Platinum Taq High Fidelity amplify region of interest (design primers, should be <1000bp):
  • Per reaction:
    • 21.5 uL Platinum Taq High Fidelity Master Mix
    • 2.5 uL of Primers from a 2 uM stock solution
    • 1 uL of the Extracted DNA from the previous step.
  • The PCR program should be
    • 2 min at 95C
    • 94C for 15
    • 55C for 15s (adjust based on Tm of primers, use 5C less)
    • 68C for 1min/kb amplicon
    • Repeat steps 2-5 35X
    • 68C for 10min

Assay

  1. To do the surveyor assay, first heteroduplexes must be made. If your mixture is expected to have a combination of hetero- and homo-duplexes already (ie is not a clonal population) you can skip step 2 and just work from the PCR product directly
  2. Combine in a new tube, amplified WT DNA (~10 uL) and the test DNA (~10 uL). As a control also make up a tube of just WT DNA.
  3. Run the heteroduplex program (10 min at 95C, followed by 1min at 85,75,65,55,45,35 then at RT).
  4. Add 1uL Surveyor Nuclease S to the mixture
  5. Add 1uL Surveyor Enhancer S to the mixture
  6. Digest at 42C for 60min
  7. Analyse on a 2% agarose gel.