Surveyor Assay
Adapted from IDT surveyor assay protocol and F A Ran et.al.,Nat. Prot. 2013.
Overview:
Ø DNA isolation
Ø PCR amplification of targeted region from genomic DNA from both untargeted and targeted cells
Ø Heteroduplex annealing of PCR amplified wild type and mutant (targeted) fragments
Ø Surveyor digestion and Visualization
DNA isolation
1) Centrifuge down 105 cells in a 1.5ml Eppendorf and add 200 µl of QuickExtract™ DNA Extraction solution.
2) Vortex mix for 15 seconds.
3) Incubate at 65°C for 6 minutes.
4) Vortex mix for 15 seconds.
5) Transfer the tube to 98°C and incubate for 2 minutes.
6) Store the DNA at –20°C, or at –70°C for long term storage.
7) Use 2µl (upto5 µl) of the extracted DNA for each PCR amplification.
Note: If using purified DNA use ~100ng of template DNA for each PCR reaction.
·
PCR amplification of fragment from the genomic DNA
Primer Design
· Primer should be designed using DNA sequence (not CDS) including introns.
· Surveyor primers should be designed to amplify 200–400 bp on either side of the Cas9 target (for a total amplicon 400–800 bp long) to allow clear visualization of cleavage bands by gel electrophoresis (F A Ran et al, Nat prot. 2013).
· Primers should be designed to be 18-25 nucleotides with melting temperatures of ~60 °C. Primers with lower melting temperature should be avoided to minimize nonspecific annealing and formation of primer dimer.
PCR reaction
8 PCR mix (50 µl)
Q5 polymerase Buffer 10µl
Q5 High GC enhancer 10 µl
dNTP mix 2 µl
Q5 polymerase 0.5µl
Primer F 2 µl
Primer R 2 µl
Template (~100ng, if using purified DNA or 2-3µl of Quick extract isolated DNA)
H2O to 50 µl
9) PCR condition
9) PCR condition
980C- 20sec
600C- 30sec 30cycles
720C- 15sec*
720C- 2min
40C- pause
Important Notes:
*15 sec extension works well for ~570bp fragment of RNF43-8, for longer fragments it can be increased. New IDT protocol recommends 30seconds/250bp.
Before proceeding to next step:
10) Run 5 µl on 2% agarose gel and confirm that there is a single band of desired size.
11) Purify the PCR product and quantify using nanodrop, Yield of the PCR should be more than 25ng/µl, if less repeat and standardize the PCR reaction.
Heteroduplex Annealing
Wild type and targeted DNA are mixed and hybridized to form heteroduplexes. In cases where there is heterogeneous DNA population (from non-selected/ partially selected cells) mixing of test DNA with wild type DNA is not required.
DNA should be in the range 25–80 ng/ µl (ideally 50 ng/ µl); ~700ng of DNA should be used for heteroduplex annealing.
Appropriate negative (homoduplex) and positive (known heteroduplex/DNA sample with mismatch) controls should also be annealed alongside.
12) Mix the reaction as follows in 0.2ml PCR tube:
10X Pfu PCR buffer- 2 µl
PCR product (25-80ng/ µl) - 18 µl
13) Anneal in thermal cycler using following condition.
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Surveyor Digestion and Visualization
14) Setup Digestion reaction as follows in 0.2ml PCR tube:
It Can be prepared as master-mix to reduce the number of manipulations. This mixture should be used immediately after preparation and should not be stored  15) Incubate at 420C for 60min. 16) Add 1/10th volume of Stop Solution and mix. Store the digestion products at –20°C if not analyzed immediately. |
Visualization :
17) Run the digested product on 2% agarose gel at 100volts in fresh TAE buffer.
18) Gel should be imaged once the loading dye has crossed 1/3rd of the gel* and then every 5-10mins to get best image.
*Smaller products <100bp are detectable early during the electrophoretic run, and bigger fragments resolve later.
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