Hybridization and Detection of Dig-Labeled Probes

NOTE: These protocols have been optimized for hybridizations in siliconized glass bottles (e.g. Robbins
Scientific Corp. or similar) and in polypropylene Corning tubes; handle membranes with extreme care
by their top or bottom edges using clean filter forceps (Nalgene) and make sure that they never dry.
1. Prehybridize blots for 1-3 h (at least 2 h the first time) in an oven at 65•C, in a tray with enough HYB solution
to cover well all the blots. The HYB solution used for pre-hybridization can be stored frozen or at 4•C and be
re-used three to four times or until precipitated material will not go into solution upon heating.
2. Roll wet membranes on a thick glass pipette on top of a flat, clean surface wetted with some of the HYB
solution from the tray, and insert them into clean hybridization bottles. Make sure that they do not roll on
themselves upon rotation in the oven (ìtacoî syndrome, check direction of rotation of rotisserie mechanism),
and avoid the formation of air bubbles or any drying of the membranes. You can place up to five 500 cm2
membranes in one bottle. Smaller membranes can be placed in 15 or 50 ml Corning polypropylene tubes
which can be fitted into sections of common PVC tubing of the right diameter, and long enough to take two
tubes each.
3. Add 2-3 ml/100 cm2 HYB solution for first blot and 1 ml more for each extra 500 cm2 membrane; in the case
of small membranes in tubes, adjust the volume accordingly. The HYB solution should contain at least 100
ng/ml of 2.5-5% Dig-labeled probe [denature probe by heating at 95•C for 10 min and quenching on ice]. If
HYB solution containing probe has been previously used and stored frozen, thaw and denature for 20 min at
NOTE: After the first use, the intensity of the signal on the membrane will start to decrease; it will thus be
necessary to gradually increase the concentration of the probe in the HYB solution and/or increase
the concentration of AMPPD (see below), with each re-use.
4. Hybridize for 15-18 h (overnight) at 65•C in bottles in hybridization oven.
5. Remove membranes from bottle(s) and wash together in trays of adequate size with shaking as follows:
NOTES: HYB solution containing probe may be saved at -30•C for re-use.
Clean hybridization bottles immediately to avoid formation of HYB residues.
2 x 5 min                               0.15X SSC, 0.1% SDS                    RT                      0.5 ml/cm2
3 x 15 min                             0.15X SSC, 0.1% SDS                   65•C                   0.5 ml/cm2
OR, for lower stringency,
3 x 15 min                             0.15X SSC, 0.1% SDS                    RT                      0.5 ml/cm2
1 x 15 min                             0.15X SSC, 0.1% SDS                    50•C                  0.5 ml/cm2
It is essential that the wash temperatures be monitored to make sure that the above treatments are respected
consistently; undue lowering of the temperature or shorter treatment times may result in higher background
noise and less predictable results.
6. Rinse membranes in Buffer 1 at RT*.
7. Incubate membranes in Buffer 2 for 30 min at RT with shaking (5 ml/100 cm2).
8. Incubate membranes in fresh anti-Dig solution (5 ml/100 cm2) for 30 min at RT with shaking; this solution
may be re-used in the same or the next two days of first use. [Centrifuge anti-Dig immediately prior to use and
carefully pipet desired amount].

9. Wash membranes with shaking as follows:
3 x 10 min                          Buffer 2                           RT                              0.5 ml/cm2
3 x 10 min                          Buffer 1                           RT                              0.5 ml/cm2
1 x 5 min                             Buffer 3                           RT                              0.5 ml/cm2 *
10. Incubate membranes in CSPD solution (5 ml/100 cm2), for 20 min at RT with shaking, preferably in the dark.
[Save CSPD solution between uses in refrigerator in a bottle wrapped in aluminum foil]
11. Remove each membrane from AMPPD tray slowly, letting solution drip off the membrane; then place, DNAside
down, on top of GladWrap (or equivalent wrapping plastic film). You can do several membranes in a row
on a long stretch of film secured on a table with tape. Place another sheet of GladWrap on top (back side of
membranes), cut GladWrap between membranes and seal edges on back side of each membrane.
12. Place membranes in cassettes and expose to XAR-5 X-ray film overnight (15-18 h).
Note that this long exposure has been sought in the development of this protocol in order to facilitate the
simultaneous handling of several dozen large membranes; in addition, it provides a natural overnight break for
the worker in charge.
13. Develop X-ray film for 6 min in GBX (Kodak) developer, rinse in H2O for 30 sec, fix in GBX fixer for 3 min,
and rinse for 3 min in running H2O.

NOTE: If signal is weak (you can at least see some faint bands), the membranes can be incubated in
higher strength AMPPD and re-exposed by starting with either Buffer 2 (Step 7) or Buffer 3 (Step
9) wash above.
14. In order to ensure a longer life of the membranes as well as a successful stripping of the probe, proceed
immediately to remove them from their plastic wrapping and immerse them in 0.1X SSC, 0.1% SDS (ìHighest
Stringency Washî) or in 2X SSC in a tray at RT. Do not allow the membranes to dry! You may keep them for
a few days in this solution at 4•C or, better still, strip them right away (see next protocol, p. 32).
15. Score your results on a ìHybridization data sheetî (example given at end of manual) for signal, lane
background, and general background using the following rating scale:
0                          1                      2                      3
none                  faint             medium        strong
This will help you keep track of probe quality and/or other problems during these steps.

HYB Solution
STOCK                                    [FINAL]                          25 ml                          50 ml                      75 ml                        100 ml                             150 ml
25X SSC                                 5X SSC                                5 ml                          10 ml                       15 ml                           20 ml                               30 ml
10% Laurylsarcosine       0.01%                               25 μl                           50 μl                        75 μl                          100 μl                              150 μl
20% SDS (good)                  0.02%                               25 μl                          50 μl                        75 μl                           100 μl                              150 μl
Blocking reagent1                 0.2%                             50 mg                      100 mg                   150 mg                        200 mg                            300 mg
(Boehringer Mannheim)     0.3%                            75 mg                      150 mg                    225 mg                        300 mg                            450 mg
1 Add after heating the solution to 65•C and checking that the pH is 7.4. We use 0.2% for maize and 0.3%
for wheat.
0.10X SSC, 0.1% SDS: Highest Stringency Wash
STOCK                             1000 ml                            2000 ml                      3000 ml                    4000 ml                5000 ml                          6000 ml
25X SSC                               4.0 ml                                8.0 ml                        12.0 ml                       16.0 ml                  20.0 ml                           24.0 ml
20% SDS (cheap)             5.0 ml                              10.0 ml                        15.0 ml                      20.0 ml                   25.0 ml                          30.0 ml

0.15X SSC, 0.1% SDS: Higher Stringency Wash
STOCK                          1000 ml                    2000 ml                3000 ml             4000 ml                      5000 ml                     6000 ml
25X SSC                           6.0 ml                       12.0 ml                  18.0 ml               24.0 ml                        30.0 ml                       36.0 ml
20% SDS (cheap)         5.0 ml                       10.0 ml                  15.0 ml               20.0 ml                        25.0 ml                       30.0 ml

0.20X SSC, 0.1% SDS: High Stringency Wash
STOCK                         1000 ml                      2000 ml                3000 ml             4000 ml                      5000 ml                     6000 ml
25X SSC                          8.0 ml                         16.0 ml                  24.0 ml               32.0 ml                        40.0 ml                       48.0 ml
20% SDS (cheap)        5.0 ml                         10.0 ml                   15.0 ml              20.0 ml                         25.0 ml                      30.0 ml

Buffer 1
STOCK                                                       [FINAL]                      500 ml                        1000 ml                          2000 ml                            4000 ml
1 M Tris-HCl, pH 7.5 0.01 M            5.0 ml                         10.0 ml                         20.0 ml                            40.0 ml
5 M NaCl                                                  0.15 M                          15.0 ml                         30.0 ml                            60.0 ml                          120.0 ml

Buffer 2
STOCK                                                        [FINAL]                            500 ml                     1000 ml                            2000 ml                             4000 ml
1 M Tris-HCl, pH 7.5 0.01 M              5.0 ml                              10.0 ml                      20.0 ml                              40.0 ml
5 M NaCl                                                    0.15 M                              15.0 ml                       30.0 ml                              60.0 ml                           120.0 ml
Blocking reagent maize                          0.1%                            500.0 mg                1000.0 mg                       2000.0 mg                      4000.0 mg
(Boehringer Mannheim                 wheat 0.2%                    1000.0 mg                2000.0 mg                       4000.0 mg                      8000.0 mg
To dissolve the blocking reagent, first heat the solution to 65•C before adding it (Beware: never heat
solution already containing blocking reagent in microwave). You may prepare this solution up to a day
before use but make sure to use it at room temperature.

Buffer 3
STOCK                                                    [FINAL]                                        100 ml                    200 ml                        400 ml                             500 ml
1 M Tris-HCl, pH 9.5 0.10 M          10.0 ml                                       20.0 ml                  40.0 ml                       50.0 ml
5 M NaCl                                                  0.10 M                                          2.0 ml                     4.0 ml                          8.0 ml                            10.0 ml
Autoclave solution before use or use autoclaved stocks and ddH2O.

Anti-Dig (1:15000)
Buffer 2 + 1 μl/15 ml anti-Dig (Anti-digoxigenin-AP, Boehringer Mannheim, Cat. # 1093274, 150
Units/200 μl)

CSPD Solution (2 μl/ml)
Buffer 3 + 2 μl/ml CSPDD (Tropix , Cat. No. CD100R, 10 mg/ml)
NOTES: The concentration of CSPD can be increased after few uses and the signal decreases with each reuse
of the membrane.
Diluted CSPD solution should be stored at 4•C in a bottle wrapped in aluminum foil. The solution
can be re-used several (5-10) times and can be filter sterilized every few uses to avoid

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