incubate gel

(not necessary if gels are run in TBE or TPE without ethidium bromide or other intercalating agents)

- 3x 20' 1xTBE (or TPE)

- (optional) 10' ethidium bromide, shoot a pic

- (3x 10' water)

- 20' denaturation solution

- 25' neutralization solution

- 10' 20x SSC

blot

- sandwich (from BOTTOM to TOP):

 - 3 whatmann papers (soaked in 20x SSC)
 - gel
 - nylon, hybond, whatever (soak membrane in water, then add 4 volumes of 20x SSC)
 - 3 whatmann papers (soaked in 20x SSC)
 - pile of paper
 - 1 kg weight

NO BUBBLES !!! (remove by rolling a pipet over the gel/membrane/paper)

- pull DNA up for 5 hours or more (like o/n, weekend) with 20x SSC

- 90'' X-link on transilluminator (or normal stratalinker X-link)

block, probe, detect

- prehybridize 4 hours or more at 65 C

- (DIGed plasmid) probe 5 hours to o/n

washing away unbound label: optimal times and temps vary with different probes

- 10' 65 C 1x SSC, 0.1% SDS

- 10' 65 C 0.5x SSC, 0.1% SDS

- 10' 65 C 0.1x SSC, 0.1% SDS

- 0.1x SSC, cool from 65 C to rt

- 5' 0.3% tween20/TBS

- 30' 1% BBR/TBS (BBR = boehringer blocking reagent)

- add antiDIG-AP, 1:10k, 30'

- wash 3x 10' in 0.3% tween20/TBS

- 5' AP buffer

- 5' CSPD

- film or scanner

solutions

10x TPE

151.5 g Tris
71.25 g H2PO4 (=43.75 ml from an 85% solution)
9.3 g EDTA
water to 2.5 l

20x SSC

3M NaCl, 0.3 M Na-citrate (pH=about 7)

880 g NaCl and 440 g sodium citrate in 2.5 l water

10x TBS

200 mM Tris pH 7.5
1.5 M NaCl

AP buffer (DIG3 from Boehringer DIG kit)

100 mM Tris pH 9.5
100 mM NaCl
(optional: 5 mM MgCl2)

prepare as 10x stock solution - do not add MgCl2 to 10x stock solution, it will precipitate

denaturation

50 g NaOH
219 g NaCl
water to 2.5 l

neutralization

302.7 g Tris
219 g NaCl
water to about 2 l
HCl to pH 7
(takes >100 ml 37% HCl)
water to 2.5 l

prehyb

5x SSC
1% BBR
0.2% SDS,
0.1% lauroylsarcosine