IN VITRO FOOTPRINTING METHODS
Preparation of labeled probes:
There are several choices available in the labeling of DNA termini for subsequent footprinting procedures-- here are two that work well:
The old way: Gel purification.
1. a.) A convenient subclone of the HSP70 promoter region is pK!3 which contains HSP70 promoter sequences from -188 bp to -4 bp. To single-end label the DNA 50 μg of pK!3 was digested with either Sal I (-188 bp) or Hind III (-4 bp). All of the DNA was treated with CIP as described by the manufacturer and the CIP was inactivated by heating to 65°C in the presence of 12.5 mM EGTA and organic extraction. After ethanol precipitation the DNAs were resuspended at 1 μg/μl.
b.) 5 μg of plasmid DNA linearized at either the Sal I or Hind III site was labeled with 10 units of T4 DNA kinase or E. coli Klenow fragment as described in Maniatis except that the reaction was performed in 10μl. Generally, 150-200 μCi were used per labeling reaction (5 μg of plasmid). Labeling proceeded for 30 min at 37°C and was terminated by the addition of EDTA and dilution to 100 μl with TE.
c.) The labeled DNA was then purified by spin chromatography over a Sephadex G-50 column. The DNA was then digested with a four-fold excess of the complementary enzyme (Sal I or Hind III whichever was appropriate for release of the single-end labeled fragment).
d.) The entire digest ("120 μl) was loaded onto a 1.5 mm 4% 19:1 native acrylamide gel prepared in 0.5X TBE (use gel-shift size plates) and poured with the large 10 well BioRad prep comb. The entire 120 μl + tracking dye was loaded into one lane (yes, it's possible). Electrophoresis was at 150 - 200 V for approximately 2 - 3 hours depending on the size of the fragment.
e.) To visualize the position of the radioactive band of interest the gel was taken apart leaving the gel on one of the glass plates. It was quickly covered with SaranWrap and another clean glass plate (work behind a shield as there is a fair amount of exposure at this point). Take the covered gel and 8 x 10 X-ray film to the darkroom. Carefully overlay a sheet of film onto the gel (on top of the SaranWrap)-- I always line up the edges of the film with two edges of the glass plate -- put the clean glass plate back on for two minutes. Remove film and process. The desired band should be readily visible -- a dark black if you've done well.
f.) While the gel is still on the plate place the film underneath and align the film so that it indicates the position of the band in the gel (work behind shield). Use a clean razor blade to excise the band (remove SaranWrap before crushing). Place the gel slice in a 1.5 ml eppy tube and grind the gel fragment into a paste (I use a p200 pipette tip). Add 400 μl of 0.5 M Ammonium Acetate, 1 mM EDTA to the ground gel and incubate overnight with shaking at 37°C.
g.) Recover eluted DNA by centrifugation at high speed for 5 min. Remove as much of the supernatant as possible to a new tube. Add 150 μl of the elution buffer to the crushed gel pellet and vortex, spin again and combine the supernatant with the first. Using the Geiger counter you should find that 80-90% of the radioactivity is in the recovered supernatant. If the fragment is longer than 300 basepairs then recovery will decline.
h.) Spin the supernatant again for 15 min at high speed. This helps to remove small pieces of acrylamide. Transfer supernatant to new tube and ethanol precipitate with 3 volumes of 95%. No salt or carrier is necessary. After centrifugation the DNA was resuspended in approximately 100 μl and 0.5 to 1.0 μl were used for each footprinting reaction (5 - 10 fmoles, 10, 000 - 20,000 cpm). Purine ladders for markers were prepared as described previously (Maxam and Gilbert, 1980) with formic acid treatment and piperidine cleavage.
The newer (not necessarily better) way:
2. a.) The polymerase chain reaction can be used to prepare radioactive probes of high specific activity. Although any two primers could be used in this example the T7 and T3 promoter primers complementary to Bluescript KS- were used to prepare single end-labeled probes. The T7 primer was labeled to high specific activity with T4 DNA kinase as above except 1 mCi of g-ATP was used per 1 μg of primer as well as 20 units of T4 kinase.
