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FAST AND CLEAN GRUNSTEIN HOGNESS PROCEDURE

(by Jerry Koudelka)

  1. 1. Patch individual colonies onto LB plates with antibiotics and nitrocellulose filters placed on LB plates. Grow at 37 deg.C. 3 hours to overnight. (I usually do it 3 hours)
  2. Take filters, and place on 1 sheets of 10mm Whatman paper that you have presoaked with 10% SDS. Leave for 5 min.
  3. Place filters on 1 sheets Whatman paper presoaked with denaturing solution (0.5M NaOH, 1.5M NaCl) for 5 min.
  4. Place filters on 1 sheets of Whatman paper presoaked with renaturing solution (0.5M Tris, pH 8.0, 1.5M NaCl) for 5 min.
  5. Place filters on 1 sheets of Whatman paper presoaked with 2X SSC or SSPE. Recipes for these are in Maniatis or Red Book. Let filters sit for 5 min.
  6. Dry filters at room temp. 15-30 min.(ignore the part in Maniatis about drying for 1-2 hours).
  7. Bake in 80 degree oven for 1 hour between two sheets of Whatman paper. . While filters are baking, prewarm 6X SSC to 65 degrees C and warm up one of the warm air shakers.
  8. Put filters in 10 ml petri dishes (one filter per dish) and prehyb using 6X SSC or 6X SSPE for 5 min at 65 degrees C.
  9. Then remove the prehyb and add probe which is in 6XSSC or 6X SSPE in a final volume of about 5 ml per petri dish. Put in 65 degree warm air shaker for 1/2 hour. Do longer if you are unsure everything is up to 65 degrees.
  10. Remove and let stand on your bench behind a shield until everything comes to Room Temp. This is an important step and is probably when the actual hybridization occurs (like hybridizing oligos).
  11. Wash with 6X SSC or 6X SSPE two times at room temperature, 10 min each time. Do a quick exposure, about 45 min at room temp. This is important because it will give you the patch pattern which is necessary for later identifying your positives. Most important: EXPOSE BY PUTTING FILTERS ON A PIECE OF WHATMAN PAPER THAT IS SOAKED IN 6X SSC. If you donŐt do this when you try to peel the saran wrap off it will pull up the colonies. Place saran wrap over filter, then film, then intensifying screen.
  12. After first exposure, put filters in 65 deg. shaker with buffer starting at about 42 degrees. Monitor every 20 min or so. When temp of buffer (not of shaker) gets to about 50-55 you should notice a big drop in counts (From about 2 to 0.2-0.5 on counter in 234 set on 1X). At this point remove filter and expose as before, this time for about 2-6 hours on wet Whatman paper.
  13. You may want to do additional washes at 65 degrees or with lower salt to improve signal to noise ratio. Experience is that if you wash overnight with 6X SSC at 65 degrees, then you will need to do an overnight exposure at -70 degrees C (which is really pretty good).
  14. Note: The above conditions are for using a kinased 30-mer as probe. I used 100 picomoles of oligo, and 2.5 ul of 3000 specific activity g 32-P, in a 25 ul kinase reaction. This is plenty for four filters of 50 patches each. You do not have to remove unincorporated nucleotides before adding to filters (Neat Huh!)