Labeling/Hybridizing/Washing

1. Prehybridizing
-Thaw 32P in isotope hood.
-Place membrane in hybridization tube.
-Add 50 ml of prehyb buffer to each tube.
-Set oven to 650C.
-Prehybridize membranes for at least 6 hr (or overnight).

Prehybridization buffer:    (for 4 probes)
125 ml	ddH2O
  60 ml	20X SSPE
  10 ml	100X Denhardt's solution
    5 ml	20% SDS
    1 ml 	salmon sperm DNA (10 mg/ml) (boil for 5 min before adding).
      200 ml

2. Probe Labeling
a. Heat water in a beaker to boiling on a hot plate.
b. Labeling mixture:
DNA			  1.0 µl (20-50 ng)
ddH2O		  4.0 (l (H20 & probe total vol. of 5(l)
Oligo primers		  1.0 µl
dNTPs (5mM)	  	  1.5 µl
Klenow polymerase	  1.0 µl
10X Klenow buffer	  1.5 µl
dCTP 32P		  5.0 µl
			15.0 µl
c. Combine H2O and DNA in a 1.5 ml microfuge tube, boil for 4 min, and place directly on ice.
d. Add oligo primer, dNTPs, 10X buffer, Klenow enzyme, and 32P.  Mix, spin briefly, and allow reaction to go overnight at room temp or place at 370C for 2 hr.

3. Preparing the  column
a. Add glass wool to the base of a 1-ml syringe, and place in a 15-ml centrifuge tube.
b. Using a transfer pipette, fill the syringe with a solution of Sephadex G-50 in TE.  Avoid air bubbles in the column.
c. Centrifuge tubes for ~30 sec., refill tubes with fresh Sephadex G-50 solution, and centrifuge at 1,500 rpm (Damon/IEC Division Centrifuge)  for 4 min.
d. Add 200 µl of TE to each column and centrifuge for 4 min at 1,500 rpm.

4. Purifying the probe
a. Add 185 µl of TE to each probe
b. Cut the cap off of the tube and withdraw all of the probe solution from the tube with a pipetter.  Place an empty tube under the syringe and add the probe to the top of the column.
c. Spin the column for 4 min at 1,500 rpm.
d. Discard the column, remove tube with purified probe, and recap.
e. Add a cap-lock to the tube and boil for 4 min. Place directly on ice.
5.   Hybridization
Hybridization buffer: 	(for 4 probes)
25.0 ml	ddH2O
12.0 ml	20X SSPE
  2.0 ml	100X Denhardt's solution
  1.0 ml	20% SDS
  0.2 ml	salmon sperm DNA (10 mg/ml) (boil for 5 min before adding).
  2.0 g	Dextran sulfate
40.2 ml

a. Replace prehybridization buffer in each hybridization bottle with 10 ml of hybridization buffer.
b. Add probe to hybridization bottle. (NOTE: do not allow the probe to come in direct contact with the membrane when first adding the probe.  Make sure the probe goes directly into the hybridization solution in the bottle.)
c. Allow the probes to hybridize for 18-20 hrs at 650 C.

6. Membrane washing
a. Remove bottle from oven, pour solution into a radioactive waste container, remove membranes from bottle, and place them into a plastic box. Rinse membranes with 2X SSPE.  Separate membranes by placing a piece of nylon mesh (Cat. CMN-300-D, Small Parts. Inc.) between each membrane.
b. Preheat the wash solution to 650 C and add to plastic box with membranes.  Place plastic box in 650 C water bath. 
c. Wash for 30 min with 2X SSPE, 30 min with 1X SSPE, and 30 min with 0.5X SSPE.

Stock:		 1.0 L		   2.0 L              
1st: 2X SSPE		20X SSPE	100 ml		  200 ml
			20% SDS	  25 ml		    50 ml
			ddH2O		875 ml		1,750 ml

2nd: 1X SSPE		20X SSPE	  50 ml		  100 ml
			20% SDS	  25 ml		    50 ml
			ddH2O		925 ml		1,850 ml

3rd: 0.5X SSPE	20X SSPE	  25 ml		    50 ml
			20% SDS	  25 ml		    50 ml
			ddH2O		950 ml		1,900 ml

d. After the last wash, place filters, DNA side up, on a piece of 3MM Whatman gel-blot paper. When the liquid has evaporated (don't let them dry too long), place in a lightweight sheet protector (treat with Sigmacote, cat. SL-2, Sigma, before use). Check signal (400-800 cpm) with a geiger counter., Put the membrane into a labeled cassette and add X-ray film in the dark room. Put into a -800 C freezer and expose (usually 4-5 days). Develop film.




7. Membrane stripping
Strip by pouring a solution of 0.5M NaOH  over the filters, and wash for 1 hour at room temperature with gentle shaking. Rinse once with distilled water and then with 2X SSPE.  Keep membrane in 2X SSPE in refrigerator until next use. 
       
Recipes for preparing solutions:

Sephadex in TE	Sephadex, Sigma Cat. S-5897
-Add 60 g of Sephadex G50 to 960 ml of ddH20 - stir for 10 min.
-Let the solution settle for 10 - 20 minutes, pour off H20.
-Repeat 3 times.
-Add TE (pH 7.6 - 8.0) to a total volume of 960 ml.
-Autoclave

100X Denhardt's
10 g Polyvinylpyrolidone       Sigma PVP-360
10 g Ficol			Sigma F-4375
10 g Albumin, Bovine 	Sigma A-9647

-Bring to a final volume of 500 ml with ddH20. Store @ 4( C.

Oligonucleotide
	Pd(N)6  5( - PO4  Na+ Salt
	Amersham Pharmacia Biotech
	Catalog #  27-2166-01
	
	1 unit = 26.5 (g
50 units ( Dilute with 500 (l TE ( 100 units/1 ml (stock)
	Make 59.6 ng/1 (l oligo
	Take 4.5 (l of 100 units/1 ml ( Add 195.5 (l ddH2O ( 200 (l of
59.6 ng/1 (l oligo

Salmon sperm DNA (10 mg/ml)
Deoxyribonucleic acid, sodium salt, USB Cat. 78019
-Add 1 g of salmon sperm DNA to 100 ml of 0.4 M NaOH, mix to dissolve.
-Boil for 30 min. in a water bath, then place on ice to cool, then cool to room temp.
-Adjust pH to 7.0 using glacial acetic acid.
-Aliquot to 50 ml tubes (15 ml to each of 7 tubes).
-Add two volumes of cold 100% ethanol and store in a -200 C freezer for at least 1            
  hour.
-Centrifuge @ 6000 rpm for 20 min.
-Wash pellet with 70% ethanol.
-Dry the pellets and combine.
-Resuspend in ddH2O (100 ml).
-Final concentration should be 10 mg/ml.
    
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