Difference between revisions of "Surveyor Assay for Genomic DNA Mutations"

Revision as of 18:01, 26 May 2015

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:
  • 22.5 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.