Day 1-Genomic Digests
Digest 10 ul of genomic DNA overnight
Day 2-Setting up Blot
1) Run gel slowly
2) Photograph gel with ruler (this will allow you to line up the markers with your bands.
3) Transfer gel to dish, cover with depurination solution and agitate. Treat until blue dye turns yellow (approximately 10-12 minutes).
4) Rinse gel with distilled water
5) Cover gel with denaturation solution and agitate. Stop 25 minutes after bromophenol blue has returned to its blue color.
6) Rinse gel with distilled water.
7) Cover gel with neutralization solution. Agitate for 30 minutes
8) Set up blot (20x SSC in resevoir and 10x SSC in wicking papers)
9) Prepare prehyb solution (0.5 M NaCl, 5% ECL blocking mix in ECL hybridization solution). Typically prepare 25 ml solution (0.73 g NaCl, 1.25 g blocking mix in 25 ml hybridization solution). Place in hybridization oven at 42degreesC.
Day 3-Taking down blot and Making the Probe
1) Carefully remove blotting paper. Mark lanes on Hybond+ with pencil
2) Rinse blot with 6x SSC and bake 2 hrs at 80degrees C.
3) Prehybe blot for 1 hr at 42 degrees.
4) While prehybing, prepare 10 ng of probe per ml hybridization fluid (250 ng).
5) Dilute DNA to 10 ng/ul (total volume=25 ul) and boil in vigorous boiling water bath for 5 minutes.
6) Immediately cool on ice for 5 minutes. Spin down liquid in tube.
7) Add 25 ul of DNA labelling reagent to cooled DNA. Mix gently.
8) Add 25 ul glutaraldehye solution. Mix thoroughly and spin briefly to collect liquid at the bottom of the tube.
9) Incubate for 10 minutes at 37degreesC (incubate 20 minutes if probe is <300 bp).
10) Put on ice until ready for use.
11) Withdraw a small amount of prehybe fluid from blot, mix with probe and add back to blot. Hybridize at 42 degrees overnight.
12) Prepare primary wash buffer and put in hybridization oven to warm to 42 degrees C.
Day 4-Processing blot
1) Wash blot for 20 minutes in primary wash buffer at 42degreesC. Discard solution and wash 2 x 10 minutes at 42C.
2) Wash 2 x 5 minutes with 20x SSC at room temperature.
3) Drain blot and add ECL detection solution. Process blot. Try 30 minutes exposure.
Depurination solution (1 liter)
250 mM HCl 20.75 ml
Denaturation solution (1 liter)
1.5 M NaCl 87.7 g
0.5 M NaOH 20 g
Neutralization solution (1 liter)
1.5 M NaCl 87.7
0.5 M Tris-HCl, pH 7.5 78.8
20x SSC (1 liter)
0.3 M Na3citrate2H20, pH 7.0 88.2
3 M NaCl 175.3
Primary Wash Buffer (1 liter)
6 M Urea 360 g
0.4% SDS 4 g
0.5x SSC 25 ml 20x SSC