Ulrike Heberlein

PREPARATION OF PROBES FOR FOOTPRINTING EXPERIMENTS

Probes are essentially labeled as for Maxam and Gilbert sequencing; one end of the DNA fragment is end-labeled with 32P-ÿATP. The general strategy is to cut the plasmid with a restriction enzyme at the site to be labeled. The 5'-phosphate is removed with alkaline phosphatase and replaced with 32P. The labeled DNA is then cut with a second restriction enzyme and the fragment of interest is isolated. It is important to make sure that a combination of digests will produce fragments that can be separated from each other by gel electrophoresis. Restriciton sites that result in a 5' overhang should be used; however, blunt ends can also be labeled with lower efficiency.

1) First digest: Digest ~5-10µg of CsCl-purified DNA (any RNA contamination will greatly reduce the efficiency of the labelling!!) with the restriction enzyme of choice in a 50µl volume. Check the digest on a gel to make sure it is complete (partial digests can cause nightmares).

2) Removal of 5' phosphate: To the above digest add: 45µl of H2O + 2.5µl of 2M Tris/HCl pH9.5 + 2.5U of calf intestine alkaline phosphatase (from Boehringer Mannheim, 1U/µl, cat. #713023). Incubate at 37šC for 30-45 min. Extract with 1 volume of 1:1 phenol/chloroform twice (this is important to get rid of all the phosphatase before labelling). Extract with 1 volume of chloroform and ethanol precipitate with 2.5 volumes of ethanol in the presence of 0.3M NaOAc. Resuspend the washed and dried pellet in 16µl H2O (this volume is based on the volumes and concentrations of reagents used in the kinasing reaction described below; adjust it if you need to).

3) Addition of 32phosphate: To the phosphatased DNA in a 16µl volume add: 2µl .i.10X .i.kinase buffer;; (0.5M Tris pH 8, 0.1M MgCl2, 50mM DTT, 1mM EDTA pH 8, 10mM spermidine; store at -20šC) + ~1µl 32P-ÿATP (~150µCi, ICN crude, 7000Ci/mmole) + 1µl polynucleotide kinase (10U/µl). Incubate at 37šC for 30-45 min. followed by 15 min. at 65šC to kill the enzyme.

4) Second digest: To the labeled DNA add: 5µl of 10X restriction enzyme buffer, 10-20U of the appropriate enzyme, and H2O to a final volume of 50µl and incubate at 37šC for 1-2 hrs. If you want to reduce the volume of radioactive liquid waste, you can precipitate the digest at this point to eliminate the non-incorporated 32P-ÿATP.

5) Isolation of labeled fragments: Separate the labeled fragments by gel electrophoresis. If you need to resolve small fragments use a 5% or 7% native acrylamide gel. Otherwise use a regular agarose gel (TAE buffer). Remember that these gels a screaming hot!!

A) Acrylamide gels: ~20cm long gels, 1.5mm thick are adequate. Add sucrose dye to the DNA, heat at 65šC for 5 min., load onto the gel and run for 2-3 hrs at ~200V. When finished, take off the top plate and cover the gel together with the bottom plate with Saran Wrap. Expose to film for 1-2 min. The wells are usually visible on the autradiogram, allowing easy alignment of film and gel; however you may want to use radioactive markers. Cut out the band of interest and electroelute in 0.5X TBE for 30-60 min. Precipitate the eluate with 1 volume of isopropanol at -20šC for 1-2 hrs. Spin for 15 min in a microfuge at ~15,000RPM. Resuspend the DNA in 50-100µl of TE and store at -20šC. About 0.1-0.5µl of this DNA should be enough for one footprinting reaction. If the concentration of DNA-binding factor to be assayed is low (as in most cell or tissue extracts) the minimum amount of probe, hopefully of high specific activity, necessary to be detected in an overnight exposure should be used. 1µl of labeled probe should give 50-200 cps on a minimonitor.

B) Agarose gels: Run a 0.8-1.2% TAE/agarose gel, stain with ethidium bromide, cut out desired band and isolate the DNA using Gene-Clean. (I presume that low melt agarose would also work, but I have never tried it.) Resuspend and store as above.

Purine cleavage sequence markers
(for footprinting experiments):
€Take 8µl of footprinting probe (~80,000 cpm Cerenkov) labelled as described.
€Add 5µl of 1mg/ml sonicated calf thymus DNA and dry in the speed-vac.
€Add 6µl of 2% formic acid (diluted fresh from 88% Malinckrodt stock stored at 4šC)
€Incubate at 37šC for 12 minutes and put on ice immediatly. This time is optimal to obtain an evenly cleaved ladder (~1 hit per molecule) of a 300bp long probe.
€Speed-vac for 2 hrs.
€Add 100µl of a 1:10 dilution of piperidine (Sigma, prepared fresh in the hood!!).
€Incubate at 92šC for 20 minutes. Put a weight on top of the tube(s) or they will pop open and release very toxic piperidine.
€Speed-vac overnight.
€Add 50µl ddH2O, dissolve and speed-vac until dry.
€Resuspend in 20µl of 0.1N NaOH + 40µl formamide dye.
€Use 1-5µl for each lane.