Surveyor Assay for Genomic DNA Mutations: Difference between revisions

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__NOTOC__

[[ Category: CRISPR ]]

[[ Category: Molecular Biology ]]

[[ Category: Cloning ]]

[[ Category: Genotyping ]]

==Materials==

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===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:

* Place in PCR tube and digest using '''quick-extract''' PCR program:

** 65C 10 minutes

** 68C 15 minutes

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# 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).

# 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.

@@ Line 1: / Line 1: @@
 __NOTOC__
+[[ Category: CRISPR ]]
+[[ Category: Molecular Biology ]]
+[[ Category: Cloning ]]
+[[ Category: Genotyping ]]
 ==Materials==
@@ Line 11: / Line 15: @@
 ===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:
+* Place in PCR tube and digest using '''quick-extract''' PCR program:
 ** 65C 10 minutes
 ** 68C 15 minutes
@@ 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
 # 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).
+# 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.