Difference between revisions of "Surveyor Assay for Genomic DNA Mutations"
From Bridges Lab Protocols
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__NOTOC__ | __NOTOC__ | ||
| + | [[ Category: CRISPR ]] | ||
| + | [[ Category: Molecular Biology ]] | ||
| + | [[ Category: Cloning ]] | ||
| + | [[ Category: Genotyping ]] | ||
==Materials== | ==Materials== | ||
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===DNA Extraction=== | ===DNA Extraction=== | ||
* Aspirate media and add 50 uL QuickExtract Reagent to a 24 well of cells. No need to wash cells. | * 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 | + | * Place in PCR tube and digest using '''quick-extract''' PCR program: |
** 65C 10 minutes | ** 65C 10 minutes | ||
** 68C 15 minutes | ** 68C 15 minutes | ||
| Line 20: | Line 24: | ||
* Using Platinum Taq High Fidelity amplify region of interest (design primers, should be <1000bp): | * Using Platinum Taq High Fidelity amplify region of interest (design primers, should be <1000bp): | ||
* Per reaction: | * Per reaction: | ||
| − | ** | + | ** 21.5 uL Platinum Taq High Fidelity Master Mix |
** 2.5 uL of Primers from a 2 uM stock solution | ** 2.5 uL of Primers from a 2 uM stock solution | ||
** 1 uL of the Extracted DNA from the previous step. | ** 1 uL of the Extracted DNA from the previous step. | ||
| Line 34: | Line 38: | ||
# 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 | # 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 | ||
# 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. | # 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. | ||
| − | # Run the | + | # Run the '''heteroduplex''' program (10 min at 95C, followed by 1min at 85,75,65,55,45,35 then at RT). |
# Add 1uL Surveyor Nuclease S to the mixture | # Add 1uL Surveyor Nuclease S to the mixture | ||
# Add 1uL Surveyor Enhancer S to the mixture | # Add 1uL Surveyor Enhancer S to the mixture | ||
# Digest at 42C for 60min | # Digest at 42C for 60min | ||
# Analyse on a 2% agarose gel. | # Analyse on a 2% agarose gel. | ||
Latest revision as of 20:02, 13 July 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:
- 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
- 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
- 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.
- Run the heteroduplex program (10 min at 95C, followed by 1min at 85,75,65,55,45,35 then at RT).
- Add 1uL Surveyor Nuclease S to the mixture
- Add 1uL Surveyor Enhancer S to the mixture
- Digest at 42C for 60min
- Analyse on a 2% agarose gel.
