Cel1 enzyme

Model Organism: Worms Methods & Reagents
1

Did anyone already try Cel1 digestion to detect point mutation in C. elegans mutants ? if yes, do you know where to buy this enzyme ? i don"t want to purify it myself from Celeri :slight_smile:

thanks a lot

2

Yes, I use CEL1, and it works well. It is made by the company Transgenomic, and you can buy it directly from them or through Fisher (Cat. #706020). The manual is unnecessarily long, so I made a quick guide for myself and others in the lab. Here it is (tried to attach the formatted pdf, but won’t work)
-Hannah

Quick Guide: Genotyping with Surveyor CEL I nuclease (Fisher/Transgenomic Cat. #706020)

Use: Genotyping worms for a mutation that does not create or destroy a restriction site.

Background: Surveyor CEL I nuclease cleaves single base pair mismatches in heteroduplex DNA. This nuclease can therefore be used to distinguish wildtype animals from animals heterozygous for a mutation of interest. Mutant homozygotes can also be identified using this nuclease, although two cleavage reactions must be performed in parallel: one reaction of the putative mutant homozygote alone, and one in which PCR product from the putative mutant is mixed with PCR product from a wildtype animal.

Components provided by manufacturer: Nuclease S; Enhancer S; 150 mM MgCl2; Stop Solution

  1. Obtain a ~300-1000 bp PCR product surrounding your mutation of interest. If necessary, perform nested PCR to obtain a robust product.

  2. Denature and re-anneal your PCR product. Total reaction time: ~ 20 min.
    95ËšC 10 min
    95ËšC to 85ËšC (-2.0 ËšC/s)
    85ËšC 1 min
    85ËšC to 75ËšC (-0.3 ËšC/s)
    75ËšC 1 min
    75ËšC to 65ËšC (-0.3 ËšC/s)
    65ËšC 1 min
    65ËšC to 55ËšC (-0.3 ËšC/s)
    55ËšC 1 min
    55ËšC to 45ËšC (-0.3 ËšC/s)
    45ËšC 1 min
    45ËšC to 35ËšC (-0.3 ËšC/s)
    35ËšC 1 min
    35ËšC to 25ËšC (-0.3 ËšC/s)
    25ËšC 1 min
    4ËšC
    Hold at 4ËšC until the next step.

  3. Perform the nuclease reaction. Nuclease activity is affected by components of the PCR buffer; thus, the reaction mix depends upon the polymerase used during PCR.

Roche Taq or Takara Ex Taq
8 ul re-hybridized PCR product
0.8 ul 150 mM MgCl2
0.5 ul Nuclease S
0.5 ul Enhancer S

Phusion with HF Buffer
8 ul re-hybridized PCR product
1.0 ul Nuclease S
1.0 ul Enhancer S

Incubate 42˚C for 1 hour. (Longer incubations should not be performed because the nuclease has 5’-exonuclease activity that will eventually chew up your DNA.)

  1. Run the nuclease reaction on an agarose gel. Heterozygotes should show cleavage of ~50% the product. To identify mutant homozygotes, perform two reactions: one of the mutant alone, and one of a 1:1 re-hybridized mixture of wildtype PCR product and PCR product from the putative mutant. Mutant homozygotes will show cleavage in the 1:1 mixture but no cleavage alone.

Notes: 1) I have reduced the amount of nuclease and enhancer recommended by the manufacturer by half because these components are expensive. Half works fine if you have a moderate-to-strong PCR product. If your PCR product is faint, you may need to double the nuclease and enhancer. 2) The manufacturer recommends adding 0.8 ul Stop Solution to terminate the nuclease reaction. I never do this. 3) The manufacturer claims that you should store your nuclease reaction at -20ËšC rather than 4ËšC. I have stored my nuclease reaction overnight at 4ËšC without a problem.

3

Hi Hannah,

thanks a lot. this is great, and exactly what I need.

best
Marie