1) Turn on 37O Water Bath, OVEN, and Hybridization Chamber to 65O

1) Turn on 37o water bath, OVEN, and hybridization chamber to 65o. 2) Look at gel in UV to make sure RNA transferred through to the blot. 3) Label blots with grease pen. 4) Wash Blot with 1x phosphate buffer for 5mins. 5) Bake blot in 80o oven. 6) Put 50ng of probe at bottom right corner of blot as a control. 7) X-link blot: press power, auto x-link, start, then wait to 0. (can store at 4o or ready for hybridization) 8) Cut blot as necessary. 9) Make fresh hybridization buffer (6ml/techne bottle) and add to Techne bottles. 10) Heat 10mg/ml herring sperm (hs) DNA @ 100o for 10mins then put on ice for 10mins. 11) Add 50uL of herring sperm for every 6mL of hybridization buffer. 12) Heat in hybridization chambers. 13) Rinse blot in 2x SSC. 14) Place blot in hybridization bottle. Wait 2hr before adding probe. 15) Prepare enough probes for how many blots. a) Use 100ng of linearized DNA for a probe… make final volume to 11uL with RNASE free H20. Place in 100o heat block for 10mins, start NTP mix immediately (part b), then spin down probes and place probes on ice for 5 mins after heating. b) Make 3uL Nucleotide Mix (ATG) from Roche random primed DNA labeling kit --- C is the radioactive label. Use 1uL of tube # 2, 4, 5 c) Add 2uL of tube #6 (random primers) d) Add 1uL of Klenow from NE Biolabs (tube #7) e) Immediately add 3uL of 32P-dCTP (final volume of rxn is 20mL) 16) Add dNTP mix into probes 17) Mix and spin briefly 18) Incubate at 37o for 30-45 mins. (start step #21 5mins before rxn ends) 19) Add 2ul of .2M EDTA pH 8.0 to inactive Klenow. 20) Add 28uL of RNase free dH20. 21) Prepare G-50 micro column. Vortex G-50 cartridge, loosen cap and place cartridge in 1.5mL eppendorf tube. Spin 3000 for 1min. Discard SN and spin again. 22) Place G-50 in new 1.5mL eppendorf tube and apply the deactivated probe mixture (50uL) in the center of the column matrix. 23) Spin at 3000rpm for 2mins. Make sure radioactive probe is clear and not pink. 24) Test radioactivity with Geiger counter. 25) Heat probes for 5min at 100o. 26) Incubate on ice for 3mins. 27) Spin briefly. 28) Add into Techne bottle (remember to label bottles). 29) Incubate O/N at 65o in hybridization chamber. 30) Next Day --- Turn on other hybridization chamber at 24o RT. 31) Quick wash Techne bottle with 2x SSC ( count 6secs). Remember to readjust SSC clamps 32) Wash again with 2x SSC for 30min at 24o hybridization chamber. 33) Wash with 1x SSC for 15min at 65o. 34) Expose film. Place blot between plastic cover. Remember to include reflective magnifying sheet. Got to dark room and add film. 35) Store cassette @ -80o for 24hrs.