Surveyor Assay for Genomic DNA Mutations: Difference between revisions
wrote initial protocol |
added categories |
||
| (One intermediate revision by the same user not shown) | |||
| Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
[[ Category: CRISPR ]] | |||
[[ Category: Molecular Biology ]] | |||
[[ Category: Cloning ]] | |||
[[ Category: Genotyping ]] | |||
==Materials== | ==Materials== | ||
| Line 11: | Line 15: | ||
===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. | ||