(Alonso-Stepanova Lab protocol)
This method is based on the paper in Nucleic Acid Research, 2004, Vol 32, No 8, p2632-2641, and on Chapter 13, p205-220, of the book Plant Functional Genomics from Humana Press, 2003, edited by Erich Grotewold.
To purify CELI:
1) Buy celery stems at any grocery store.
2) Extract juice from celery at 4ºC: grind in a household blender and filter the extract through a miracloth or fabric).
3) Using 1M Tris-HCl pH 7.7 and 0.1M PMSF in isopropanol, bring the extract to 0.1M Tris-HCl 0.1mM PMSF final concentration.
4) Centrifuge the extract for 20 min at 2600g at 4ºC to remove insoluble matter.
5) To the supernatant, add (NH4)SO4 crystals to reach ~25% saturation (144g per liter of extract). Add crystals slowly and with constant agitation on an ice bath or in a cold room.
6) Mix the extract for 30 minutes at 4ºC (a mild shaker will work).
7) Centrifuge the sample at 16200g for 40 minutes at 4ºC. Discard the pellet.
8) To the supernatant, add more (NH4)SO4 to reach ~80% saturation (additional 390g per liter of extract). Add crystals slowly and with constant agitation on an ice bath or in a cold room.
9) Mix the extract for 30 minutes at 4ºC (a mild shaker will work).
9) Centrifuge the sample at 16200g for 1.5 hours at 4ºC. Discard supernatant.
10) Resuspend the pellet in 1/10 volume of 0.1M Tris-HCl pH 7.7 0.1mM PMSF (1/10 of the initial juice volume obtained in step 2).
11) Dialyze the sample 4 times, 1 hour each, with 4x 1 liter of 0.1M Tris-HCl pH 7.7 0.1mM PMSF.
13) Make 50 µL aliquots and store at –20ºC.
To test activity:
– Prepare 10X CELI reaction buffer
1M MgSO4
1M HEPES pH 7.5
2M KCl
10% Triton X-100
20mg/ml of BSA
– Perform PCR separately on wild type and mutant DNA (or Col versus Ler, or mapping population), ideally to amplify a 1-2Kb fragment containing a SNP roughly in the middle.
– After amplification, mix 5 µL WT and 5 µL mutant PCR products (or use 10 µL of a potential heterozygote derectly) in a PCR tube; add a drop of mineral oil.
– In a thermocycler, heat the mixture for 10 min at 99ºC, reduce the temperature to 70ºC, and run 70 steps reducing the temperature 0.3ºC per step, keeping the samples for 20 seconds at each Tº, going from 70ºC to 49ºC.
– Upon completion, keep the mixture on ice.
– To the 10 µL sample, add [on ice!] 10 µL of the enzyme mixture containing 0.1-2 µL of CELI extract, 2 µL of 10X CELI reaction buffer, and H2O.
– Incubate for 15 minutes at 45ºC
– Place reactions on ice and run on an agarose gel.
Expected results:
