Hybridization with Minimal Hybridization Buffer on Magnagraph.

Hybridization with Minimal Hybridization Buffer on Magnagraph.

  1. Melt the frozen premade hyb mixture.
    Min Hyb Buffer Membrane (21X21 cm2) Oligo labeled DNA
    25 ml 1 1/4 X
    50 ml 4 1 X
    100 ml 8 2 X
    Membrane (13X8 cm2) ?
  2. Remove the appropriate amount of previously NaOH/boiled SS DNA after thawing.
  3. Add 1/10 volume of 1 N NaOH, voltex and let sit for 10 min for room temperature.
  4. Neutralize the base with 1/10 volume of 1.8 M Tris-HCl and 0.2 M Tris- Base sequencially. Vortex and let sit at room temperature for 10 min.
  5. Add prehybridization or hybridization solution and mix in a stir plate.
  6. Place in a 60 C water bath. Take an aliquote (usually 50 ml) into 50 ml PPT and mix with one probe which was boiled and cooled on ice. Cap the tube and voltex. Pour the probe-hyb solution into an 8X8" tupperware box.
  7. Lay the dry membranes, one at a time and one layer at a time into the solution. Tilt the box to help spread the solution. When the membrane is completely wet, add the next one.
  8. After all blots have been added, spread a layer of hybrid-probe solution over the top.
  9. Place a layer of plastic wrap directly onto the membranes to prevent evaporation. Cover with the box with a lid and incubate at 60 C overnight without shaking.

Minimal Hybridization Buffer

Conc.		Stock		1L		2L


10 %		50 % PEG	200		400


7 %		20 % SDS	350		700


0.6 X		25 X SSC	24		48


10 mM		1 M NaPO4	10		20


5 mM		0.5 M EDTA	10		20


100 µg/ml Denature Salmon Sperm	10		20


		H2O		396		792


Aliquote 45 ml into 50 ml PPT, store aat -20 C.



50 % PEG

Add 100 g PEG (Polyethylene Glycol, appr. MW 8.000) to 50 ml
H2O and stir for about one hour.  Then bring up to 200 ml with
H2O.