1) Heat Church's Buffer to 50 C, with stirring to ensure all SDS in solution.
2) Random label 75ng DNA probe as follows:
a.) Add 75ng DNA to microfuge tube, bring volume to 45µl with ddH2O.
b.) Boil DNA at 98 C for 5 minutes. Place immediatly on ice.
c.) On ice, add 6µl dATP, 6µl dTTP, 6µl dGTP, 45µl Random primer buffer, and 24µl ddH2O. Vortex to mix, centrifuge several seconds to collect.
d.) Add 15µl 32PdCTP, vortex, centrifuge, then add 3µl Klenow Fragment. Mix gently. Incubate at room temperature for one hour.
e.) Use Qiagen nucleotide removal columns (2) to recover labeled DNA. See Qiagen protocol. N.B. used two columns, eluted each with 50µl ddH2O.
3.) Pre-Wet membranes in 50 C Church's Bufffer. Taking care that both sides of the membrane fully soak in buffer. This is done by slowly lowering into buffer once each side face down.
4.) Likewise, prewet 40micron mesh filters in church's buffer. Prepare "sandwich" of membranes with mesh filters between each. Pile 10 filters each "sandwich".
5.) Place pile of 10 filters and mesh into a seal-a-meal bag. Seal end. Using pipet, or gloved hand, force all air bubbles to one corner of the bag. Make a small cut in that corner, and remove all air bubbles.
6.) Add Church's buffer to the membranes by pipeting into the cut in the bag. Add enough so that there is approximately 20ml of buffer present.
7.) Boil the labeled DNA probe from #2 above, 98 C for 5 minutes. Add 1ml Church's buffer per tube of 50µl eluted DNA. Using a 5ml Syringe fitted with an 18 gauge needle, inject the labeled DNA solution into the bag--carefully so not to introduce any bubbles.
8.) Reseal the bag.
9.) invert the bag SEVERAL times to ensure the mixing of the radioactive probe.
10.) Place the bag into a tupperware container, with H2O, Cover tupperware, place in H2O bath set at 50 degrees. Place lead weight on top of Tupperware and mix gently overnight.
11.) The following day, reheat church's buffer to 50 C. Prepare 2xSSC, 0.1%SDS buffer and heat to 65 C.
12.) remove membranes from seal-a-meal bags, place each "sandwich" into individual tupperware containters. Rinse 2-3 times in Church's buffer. TAKE CARE IN DECANTING ALL BUFFERS AS THEY WILL CONTAIN RADIOACTIVE PROBE.
13.) Add enough Church's buffer to cover membranes, place lid on Tupperware, and place into 50 C bath. Wash with agitation for 30 minutes.
14.) Turn H2O bath temperature control to 65 C.
15.) Repeat wash in fresh Church's Buffer.
16.) Wash in 2xSSC, 0.1%SDS again, 30 minutes. Bath should now be at 65 C (or approaching)
17.) Check radioactivity of Membranes. Use own judgement if additional washes needed.
18.) Wrap membranes in Cellophane wrap, place in Autorad cassette, and autorad for desired time.
Bound DNA probe removal
19.) Remove bound probe by Washing membranes at 95 C for several minutes in 0.2XSSC. This is also an efficient way to wash the mesh filters.
20.) Membranes can be stored at 4 C, in petrie dish. Protect from drying by parafilming.

CHURCH'S BUFFER:
800ml ddH2O
10 g BSA (final 1%)
35.5g Na2HPO4 (final 0.5M)
30.0g NaH2PO4 (final 0.5M)
Mix these into solution. (may require several hours) Adjust pH to 7.0
2ml 0.5M EDTA (final 0.001M)
70g SDS. (final 7%)
Mix with heat (50 C) to get SDS into solution.
qs. to 1000ml.

20x SSC:
3M NaCl (175g/L)
0.3M Na3Citrate"2H2O (88g/L)
Adjust pH to 7.0 with 1M HCl