Cel I Assay to Detect Non-Homologous Endjoining Repair

Protocol: Cel I Nuclease Assay


  • PCR reaction tube of potentially heterozygous DNA sample
  • PCR tubes, Pipetman, and tips
  • Crude Cel I (celery extract)
  • Microwave Oven
  • Glass beaker
  • Microcentrifuge tube floating rack
  • PCR thermal cycler or other heat block set to 45C
  • Stop buffer: mix one volume of loading dye with one volume of 500 mM EDTA
  • 1.5% Agarose gel in TBE


1. Denature PCR amplified DNA:

  • Place 4 ul of PCR reaction in a PCR tube
  • Heat 200 ml of water in a glass beaker in a microwave oven to boiling Remove beaker from microwave oven
  • Immediately add PCR tube(s) in floating rack to hot water
  • Wait until water cools to room temperature

2. Cel I Treatment:

  • Remove PCR tube from floating rack
  • Spin down tube in microcentrifuge
  • Add 6 ul of crude Cel I
  • Incubate at 45C for five minutes
  • Place tube on ice
  • Add 5 ul stop buffer
  • Place tube on ice

3. Gel Analysis:

  • Load samples onto 1.5% agarose gel
  • Apply 50 V and slowly run the sample into the gel
  • Photograph gel and store image in archive


Otto EA, Helou J, Allen SJ, O’Toole JF, Wise EL, Ashraf S, Attanasio M, Zhou W, Wolf MT, Hildebrandt F. 2008. Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing. Hum Mutat. 29:418-26.

Celery Juice Extract Protocol

Kindly Provided by Susan Allen (sjallen@umich.edu)

1. Rinse and dry celery stalks. Trim away leaves and bottom white parts. Weigh trimmed celery (Actual weight_____gm)
2. In cold room set up juicer. Juice celery. (Actual volume _____ ml)
3. In cold room set up stirrer with beaker of celery juice. Adjust to FINAL conc of 0.1 M Tris HCl, pH 7.7, 100 μm PMSF

Initial vol of CJ = _____ml + Stock Tris vol _____ml + Stock PMSF vol ____ ml = Total ________ml

Stock Tris = 1M Tris HCl, pH 7.7
For 1 liter of 1 M use 121.14 g then pH to 7.7 with conc HCl
Need about 4 liters for 1 bunch of celery.
Use 10 ml stock for every 100 ml Final Volume.

Stock PMSF = 0.1 M PMSF in isopropanol ( preferably anhydrous)
For 100 ml use 1.742 g then heat @ 35ºC for approx 10 min (may need longer)
Use 0.1 ml stock for every 100 ml Final Volume.

4. Spin (Beckman J221/ Rotor JA 14/ 2600 g or approx 4000 rpm/ 20 min/ 4ºC)
5. Pellet: DISCARD, Sup: KEEP
6. Bring supernatant to 25% (NH4)2SO4.In cold room with gentle stirring add 144 g of (NH4)2SO4 per liter of sup. Mix at least 30 min.
7. Spin (Beckman J221/ Rotor JA 14/ 15,300 g or approx 10,000 rpm/40 min/4ºC)
8. Pellet: DISCARD, Sup: KEEP
9. Bring supernatant to 80 % (NH4)2SO4. In cold room with gentle stirring add 390 g of (NH4)2SO4 per liter of sup. Mix at least 30 min (or O/N).
10. Spin (Beckman J221/Rotor JA 14/15,300 g or approx 10,000 rpm/ 90 min/4ºC)
11. Pellet: KEEP (can store at –80ºC ), Sup: DISCARD SLOWLY
12. Resuspend pellet in 0.1M Tris HCl, pH 7.7, 100 μM PMSF in final vol equal to one tenth of starting volume. Keep on ice.

Pellet volume:____________1/10th starting volume_________

13. Transfer to dialysis tubing (Spectra /Por, 10,000 MWCO). Prepare tubing by soaking in H20 to remove Na Azide (change every hour – 6 changes total)
14. Dialyze in cold room against 0.1M Tris HCl, pH 7.7, 100 μM PMSF. Use 7 changes of media with the first 4 changes lasting approx 1 hour each

4 liters = 400 ml of Stock Tris
               4.0 ml of Stock PMSF
qs to 4 liters with H20

1 X 4L___________
2 X 4L___________
3 X 4L___________
4 X 4L___________
5 X 4L___________
6 X 4L___________
7 X 4L___________

15. Buffer as below then store celery juice extract (CJE) in 10 ml aliquots @ -20ºC.

For each 10 ml aliquot add to buffer: 10 μl of Triton X-100
100 μl of 1M KCl
100 μl of 1M MgCl2
100 μl of 100x BSA stock

Till, BJ et al Nuclei Acids Research (2004) Vol 32 #8, p. 2632-2641
Till, BJ et al Methods in Molecular Biology (2003) Vol 236, p. 205-220

Translate »