Troubleshooting no cutting on a surveyor assay

The gel shows one band, the same size as the uncut amplicon, in every lane you hoped would be edited. Troubleshooting no cutting on a surveyor assay starts from what the enzyme is allowed to see. Surveyor nuclease and T7 endonuclease I cleave DNA where the two strands do not match. They are not general proof that a guide RNA found its target. A blank digest is a branch point, not a verdict. The cut itself, when CRISPR-Cas9 made it, is explained in how CRISPR-Cas9 editing works in research. Naming the alleles properly is in checking whether a genome edit worked.

The heteroduplex is the substrate

Polymerase chain reaction copies whatever alleles were in the cells, and each new duplex is usually a perfect match to its own template. If the well contains both wild-type and indel alleles, or two different indels, the tube of product holds more than one sequence. Those sequences still sit in separate duplexes until you denature them and let strands reanneal. Only then do a wild-type strand and an edited strand form a heteroduplex with a bubble or a loop. That mismatch is what the enzyme cuts.

Skip the denature-anneal and a true mixture can look negative. Anneal under conditions the enzyme supplier describes for that kit, not under a temperature you remember from a different mismatch assay. Follow the manufacturer's protocol for times and temperatures. This page does not replace it.

A parental amplicon, annealed alone, should stay largely uncut if the parent is uniform across the amplicon. A known mismatched control, if you have one, should cut. Those two lanes decide whether the enzyme and the thermal step worked before any experimental lane is believed.

Branch when the experimental lane is blank

Work in an order that throws away whole classes of failure. Do not change the guide, the cells, and the enzyme in the same afternoon.

First look at the PCR, before any digest. A weak, smeared, or wrong-sized product is not a substrate. Primer-dimers and off-locus amplicons produce pretty nonsense after a nuclease. The discipline in PCR controls and contamination control applies: no-template control, and a parent lane that amplifies with the same primers. If the PCR failed, stop. Fix amplification. A digest of a failed PCR cannot tell you about editing. Gel habits that make size calls fair are in agarose gel electrophoresis for DNA.

If the PCR is a clean band, ask whether the alleles were ever different from each other. A clone in which every allele carries the same insertion will reanneal as homoduplex. The enzyme is behaving. The biology may be a homozygous edit, which is the opposite of "no editing". Mix that purified amplicon with a wild-type amplicon from the parent, then denature and anneal again. Cleavage after the mix means the clone differed from the parent and the first gel was blind to it. No cleavage after a controlled mix means the clone and the parent still look the same to this enzyme across this amplicon.

If the mix with wild type still will not cut, check the enzyme on a heteroduplex you trust. Dead enzyme, an enzyme left at the wrong storage temperature, or a buffer from another kit will spare both the control and the samples. Replace the enzyme and repeat the control. Do not conclude the guide failed while the control is also blank.

If the enzyme control cuts, the PCR is clean, and mixing with wild type still yields only the parent-sized band, the remaining branch is that this population does not contain a mismatch the enzyme can cut at a level you can see. That is compatible with a guide that never cut, with edits too rare for the assay, and with a mismatch the enzyme cuts poorly. One-base bubbles are often weaker substrates than larger loops. This is why a negative Surveyor lane is not a quantitative zero. Take the same amplicon to sequencing, as outlined in what sequence data to request after an edit, before you discard the guide.

When the gel, not the biology, hides the cut

Cleavage products have to be separable from the parent and from each other. A cut very close to one primer yields a tiny fragment and a near-full-length fragment. On a short agarose run the large piece sits on top of the uncut band, and the small piece is faint or has left the gel. Increase the resolution the way you would for any close pair of DNA sizes, and load an uncut aliquot beside the digest so you can see whether the parent band actually diminished.

Overloading hides small products under smear. Underrunning leaves everything in one smile. Photograph the marker. A cropped rectangle with no marker and no uncut lane is not a troubleshooting record.

Parental single-nucleotide differences inside the amplicon can create a heteroduplex that has nothing to do with your guide. If the parent lane, annealed alone, already cuts, you are looking at heterozygosity or a mixed template, not at an edit. Sequence the parent before you redesign the guide.

Failure modes that mimic one another

A guide that never cut, a homozygous edit, and a skipped anneal can all end as one uncut band. The branches above separate them only if each control is present. Changing three variables at once returns you to the same photograph.

Enzyme activity and PCR quality also trade places. A smear digested into a smear still looks like a smear. Clean the PCR question first. Contamination of the parent with an edited amplicon from last week makes the parent lane cut and makes every sample look edited. That is a PCR-room problem, not a hyperactive guide.

Do not use this assay as an off-target survey. It only reports mismatch inside the amplicon you chose. Other loci are invisible. A knockout claim needs the sequencing and protein logic in the companion checking article, not a percentage guessed from band brightness. Band brightness is a poor fraction. Heteroduplex cleavage rarely goes to completion, and the two product bands need not look equal.

Heat, power, and enzyme storage

Mismatch annealing is a temperature programme. A cycler that reboots halfway through a power cut has not finished the programme, even if the block feels warm when you return. Repeat the anneal and include the known heteroduplex control. In a hot room, an enzyme aliquot left in a rack while gels are poured is a common way to kill the control and the samples together. Follow the storage on the label. Humidity does not change the chemistry, but it does make labels fall off tubes. An unlabelled nuclease tube is not a control.

Reagent classes for PCR and for nucleic-acid enzymes are in the molecular biology catalogue. Match the enzyme you digest with to the instruction sheet in the box you opened.

What to send when the gel will not settle it

If the branches point to sequencing, say so with coordinates, the primer sequences, and whether the sample is a clone or a pool. The CRISPR validation sequencing enquiry reference is a prompt for that discussion. It is an independent method reference. Ask whether a quotation is possible through the quote request. The molecular biology pathway is the experimental context. This assay remains a research check. It is not a diagnostic test, and it does not alter the biosafety approval that covered the edited cells.