Surveyor Assay for Genomic DNA Mutations - Revision history

Davebrid: added categories

2015-07-13T20:02:48Z

added categories

← Older revision		Revision as of 20:02, 13 July 2015
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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 quick-extract PCR program:		* Place in PCR tube and digest using '''quick-extract''' PCR program:
	** 65C 10 minutes		** 65C 10 minutes
	** 68C 15 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		# 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 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 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.

Davebrid: changed volume of polymerase

2015-06-24T13:56:38Z

changed volume of polymerase

← Older revision		Revision as of 13:56, 24 June 2015
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	* 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:
	** 22.5 uL Platinum Taq High Fidelity Master Mix		** 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.

Davebrid: wrote initial protocol

2015-05-26T18:01:23Z

wrote initial protocol

New page

__NOTOC__

==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===
# 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.