10% SDS
7% PEG
Above is sterile-filtered.
3.6 M NaCl
0.2 M Na2HPO4
0.02 M EDTA
pH to 7.7
Put on new gloves when handling blots. Heat required aliquot of sheared salmon sperm DNA in microfuge tube at 100o C for 5 minutes then immediately quench on ice. Need enough of this DNA to yield 200 ugm/mL in pre-hybridization solution.
1. Roll dry blot longitudinally in such a way that when roll is inserted into (dry) hybridization tube, the DNA side of blot is not the side touching the glass. Use nylon mesh to separate blots from one another. Can use three blots per hybridization tube.
2. Mix appropriate amount of boiled, ice-quenched sheared salmon sperm DNA (to yield a final concentration of 200 ugm/mL) with 65o C 10% SDS, 7% PEG. Pipette into hybridization tube with blot in it. Avoid pipetting bubbles into tube. Because blots are dry, 50 mLs of this mixture are necessary for pre-hybridization.
3. Following ~ 3 hour pre-hybridization at 65o C, add to one microfuge tube no more than 1 X 106 cpm/mL for each 32P-probe used and enough human Cot-1 DNA to yield a final concentration of 200 ugm/mL in this microfuge tube.
4. Hold this mixture of 32P-probes and human Cot-1 DNA at 100 oC for 5 minutes, then immediately place in 65 oC water bath. Allow one hour for the human Cot-1 DNA to anneal to any repetitive sequences in the 32P-probes. This step will lower background on autoradiogram.
5. Drain pre-hybridization solution from blots. To 15 mLs of 65 oC 10% SDS, 7% PEG (previously held in a blue capped tube in a 65 oC water bath), add enough boiled (5 min), ice-quenched, sheared salmon sperm DNA to yield a final concentration of 200 ugm/mL in this 15 mLs. Also add the annealed human Cot-1 DNA plus 32P-probes mixture. Mix and add to hybridization tubes containing pre-hybridized blots. Hybridize overnight at 65 oC. If it looks as if blots are tightening on themselves in hybridization tube, position tube in opposite orientation in hybridization oven.
6. After hybridization, pour off hybridization solution into liquid radioactive waste container. Add washes (each 150 mL) as follows. All washes are done in hybridization tube with hybridization oven set at 65 oC.
2X SSPE, 0.1% SDS 15 minutes
2X SSPE, 0.1% SDS 15 minutes
65 oC 1X SSPE, 0.1% SDS 15 minutes
65 oC 1X SSPE, 0.1% SDS 15 minutes
(The 1X SSPE, 0.1% SDS is brought to 65 oC in a microwave oven or in a 65 oC water bath.)Autoradiography of blots:
Blot lightly on unused paper towels or Whatman 3 MM paper, remove paper, cover blot with one thickness of Saran wrap, and put blot on a piece of film with orientation markers. Smooth Saran wrap or wrinkles will show up on autoradiogram. Clean off intensifying screen with water or 95% EtOH before using. Signals are visible after an overnight exposure at -80 oC with one intensifying screen.
Michael Morley |mmorley@mail.med.upenn.edu | last updated on January 7, 2003
Copyright 2003, The Children's Hospital of Philadelphia