Cel I Assay to Detect Non-Homologous Endjoining Repair

Protocol: Cel I Nuclease Assay

Materials

  • 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

Procedure

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

 

Reference:
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.

Yield:___________
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

References:
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