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__NOTOC__
[[ Category: CRISPR ]]
[[ Category: Molecular Biology ]]
[[ Category: Cloning ]]
[[ Category: Genotyping ]]
==Materials==
===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
* Using Platinum Taq High Fidelity amplify region of interest (design primers, should be <1000bp):
* Per reaction:
** 2221.5 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.
# 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.