Detection of hybridized signals

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Method A. Chemiluminescent Detection

1. Place a piece of SaranWrap film on a bench and add 0.4 ml/filter (10 x 12 cm2) of detection reagents 1 and 2 from the ECL kit (Amersham Life Science Corporation) or 0.8 ml of the Lumi-Phos 530 (Boehringer Mannheim Corporation) to the center of the SaranWrap film.

2. Wear gloves and briefly damp the filter to remove excess washing solution and thoroughly wet the DNA binding side by lifting and overlaying the filter with the solution several times.

3. Wrap the filter with SaranWrap film, leaving two ends of the film unfolded. Place the wrapped filter on a paper towel and, using another piece of paper towel, carefully press the wrapped filter to remove excess detection solution from the unfolded ends of the SaranWrap film. Excess detection solution will most likely cause a high background.

4. Completely wrap the filter and place it in an exposure cassette with DNA binding side facing up. Tape the four corners of the filter.

5. In a darkroom with the safe light on, overlay the filter with a piece of x-ray film and close the cassette. Allow exposure to proceed at room temperature for 10 s to 15 h, depending on the intensity of the detected signal.

6. In a darkroom, develop and fix the film in an appropriate developer and fixer, respectively. If an x-ray processor is available, developing, fixing, washing and drying the film can be completed in 2 min. If a hybridized signal is detected, it appears as black band on the film.

Note: It may be necessary to expose and process multiple films until the appropriate intensity of the signals is obtained. Exposure for more than 4 h may generate a high black background. In our experience, good hybridization and detection should display sharp bands within 1.5 h. In addition, the film should be slightly overexposed in order to obtain a relatively black background that will help identify the sizes of the bands compared with the marks made previously.

Method B. Colorimetric Detection of Filters Hybridized with Antibody-Conjugated Probes

1. Following hybridization and washing, place the filter in color developing solution (10 ml/filter, 10 x 12 cm2) containing 40 ml of NBT stock solution and 30 ml of BCIP stock solution. These stock solutions are commercially available.

2. Allow color to develop in the dark at room temperature for 15 to 120 min or until the desired level of detection is obtained. Positive signals should appear as a blue/purple color.

Method C. Detection of Signals by Autoradiography

If the isotopic probes are used for hybridization, autoradiography should be employed for the detection of signals.

1. Air-dry the washed filters and individually wrap the filters with a piece of SaranWrap film and place the wrapped filter in an exposure cassette with the DNA binding side facing up. Tape the four corners of the filter.

2. In a darkroom with the safe light on, overlay the filter with an x-ray film and close the cassette. Allow exposure to proceed at -80°C for 2 h to 5 days, depending on the intensity of the detected signals.

3. In a darkroom, develop and fix the film in an appropriate developer and fixer, respectively. If an x-ray processor is available, developing, fixing, washing and drying the film can be completed in 2 min. If a hybridized signal is detected, it appears as a black band on the film.

Materials Needed

5X TBE Buffer

600 ml dd.H2O

Dissolve well after each addition. Add dd.H2O to 1 l. Autoclave.

Ethidium Bromide (EtBr)

10 mg/ml in dd.H2O, dissolve well and keep in a dark or brown bottle at 4°C.

Caution: EtBr is extremely toxic and should be handled carefully.

5X Loading Buffer 50% Glycerol 2 mM EDTA 0.25% Bromphenol blue 0.25% Xylene cyanol Dissolve well and store at 4°C.

10X Nick Translation Buffer 0.5 M Tris-HCl, pH 7.5 0.1 M MgS04 1 m M DTT

500 mg/ml BSA (Fraction V, Sigma Chemical Corporation) (optional) Aliquot the stock solution and store at -20°C until use.

Unlabeled dNTP Stock Solutions 1.5mM each dNTP

Radioactive Labeled dNTP (Commercially Available) [a-32P]dATP or [a-32P]dCTP (3000 Ci/mmol)

Pancreatic DNase I Solution

DNase I (1 mg/ml) in a solution containing 0.15 M NaCl and 50% glycerol. Aliquot and store at -20°C.

E. coli DNA Polymerase I Solution Commercial suppliers

Stop Solution

1X TEN Buffer

10 mM Tris-HCl, pH 8.0 1 mM EDTA, pH 8.0 0.1 M NaCl

Sephadex G-10, G-50 or Bio-Gel P-60 Powder Commercial suppliers

5X Labeling Buffer

0.25 M Tris-HCl, pH 8.0 (from stock solution)

25 mM MgCl2

10 mM DTT (dithiothreitol) 1 mM HEPES buffer, pH 6.6 (from stock solution)

26 A260 units/ml random hexadeoxyribonucleotides

Three Unlabeled dNTPs Solution 1.5 mM of each dNTP

Klenow enzyme

5 unit/ml, labeling grade

Specific activity 3000 Ci/mmol

TE Buffer

5X Terminal Transferase Buffer 0.5 M Cacodylate, pH 6.8 1 mM CoCl2 0.5 mM DTT 500 mg/ml BSA

ssDNA Primers

Prepared from bacteriophage M13 or phagemid, sscDNA or ssDNA isolated from DNA

Terminal Transferase

From available commercial source (Promega Inc.)

[a-32P]cordycepin-5'-Phosphate Analog

3000 Ci/mmol

Depurination Solution

0.25 M HCl

Denaturing Solution 0.5 M NaOH 1.5 M NaCl

Neutralization Solution 1.5 M NaCl 1M Tris-HCl, pH 7.5

Alkaline Denaturing Solution 3 M NaCl 0.5 N NaOH

Alkaline Transfer Solution 3M NaCl 8 mM NaOH

20X SSC Solution (1 l) 175.3 g NaCl 88.4 g Sodium citrate Adjust the pH to 7.5 with HCl.

Prehybridization Buffer for Probes Prepared with the ECL Kit

Add 0.3 g blocking reagent per 10 ml of hybridization buffer. Allow the blocking powder to dissolve in the buffer for 60 min at room temperature or 30 min at 42°C with vigorous agitation.

Hybridization Buffer for Probes Prepared Using the ECL Kit

Add probe to an appropriate volume of freshly prepared prehybridization buffer.

Prehybridization Buffer for Probes Prepared with Biotin-dUTP or DIG-dUTP

5X SSC

0.1% (w/v) N-lauroylsarcosine 0.02% (w/v) Sodium dodecyl sulfate (SDS) 1% (w/v) Blocking reagent 50% (v/v) Formamide

Dissolve well on a heating plate with stirring at 65°C after each addition. Add sterile water to final volume.

Hybridization Buffer for Probes Prepared with Biotin-dUTP or DIG-dUTP

Add denatured, biotin-dUTP or DIG-dUTP-labeled probe to an appropriate volume of fresh prehybridization buffer.

50X Denhardt's Solution

1% (w/v) BSA (bovine serum albumin) 1% (w/v) Ficoll (type 400, Pharmacia) 1% (w/v) PVP (polyvinylpyrrolidone)

Dissolve well after each addition; adjust to the final volume into 500 ml aliquot with distilled water and sterile filter. Divide the solution to 50 ml each and store at -20°C. Dilute 10-fold into prehybridization and hybridization buffers.

Prehybridization Buffer for Isotopic Probes

5X Denhardt's reagent

0.2% Denatured and sheared salmon sperm DNA

Hybridization Buffer for Isotopic Probes

5X Denhardt's reagents

0.2% Denatured salmon sperm DNA

[a-32P]-labeled DNA probe

Washing Buffer 150 mM NaCl 100 mM Tris-HCl, pH 7.5

Blocking Buffer

150 mM NaCl

Dissolve well with stirring at room temperature.

Predetection/Color Developing Buffer 0.1 M Tris-HCl, pH 9.5 0.1 M NaCl 50 mM MgCl2

NBT Stock Solution

75 mg/ml Nitroblue tetrazolium (NBT) salt in 70% (v/v) dimethylformamide

BCIP Stock Solution

50 mg/ml 5-Bromo-4-chloro-3-indolyl phosphate (BCIP or X-phosphate) in 100% dimethylformamide

Other Equipment and Supplies Microcentrifuge

Sterile microcentrifuge tubes (0.5 ml) Pipettes or pipetman (0 to 200 ml, 0 to 1000 ml) Sterile pipette tips (0 to 200 ml, 0 to 1000 ml) Gel casting tray Gel combs

Electrophoresis apparatus DC power supply

Small or medium size DNA electrophoresis apparatus, depending on samples to be run Ultrapure agarose powder Nylon membranes 3MM Whatman filters Blotting paper towels Anti-DIG-alkaline phosphatase antibodies Chemiluminescent substrates

TROUBLESHOOTING GUIDE

1. Following electrophoresis, distribution of DNA species stained by EtBr is near the very top or the very bottom in some lanes instead of a long smear ranging from the top to the bottom of the lanes. These problems are most likely caused by two factors. If genomic DNA is not completely digested, DNA molecules with high molecular weights remain near the loading wells during electrophoresis. On the other hand, if DNA is somehow degraded by Dnases, they become small fragments and are distributed at the bottom of a lane. To prevent these problems, genomic DNA should be properly digested. Ensure that DNA is handled without DNase contamination.

2. After transfer is complete, obvious DNA staining remains in the agarose gel. This indicates that transfer is not efficient. Try to set up DNA transfer carefully and allow blotting to proceed for a longer time.

3. The blotted membrane shows some trace of bubbles. This is due to bubbles formed between gel and membrane. Carefully follow instructions when assembling blotting apparatus.

4. No signal is detected at all following hybridization. This is the worst and most disappointing problem in Southern blot hybridization. Genomic DNA may not be denatured before being transferred onto the membrane, or dsDNA probes may not be denatured prior to hybridization. When this occurs, it is not surprising to see a zero hybridization signal. Keep in mind that ssDNA species are the basis for hybridization.

5. Hybridized signals are quite weak on film or filter. The activities of DNA probes may be low or hybridization efficiency is not so good. The solution to this problem is to allow hybridization to proceed for a longer time and increase exposure time for x-ray film or develop it for a longer time on filter.

6. A black background occurs on x-ray film using the chemiluminescent detection method. Multiple factors may be responsible for such a common problem. The membrane filter may have dried out during hybridization or the washing process. Excess detection solution may not have been removed prior to exposure. Exposure time may have been too long. To solve this problem, make sure that the filter is kept wet and that excess detection reagents are completely removed. Try to reduce exposure time for the x-ray film.

7. A strong purple/blue background occurs on filter using the colorimet-ric detection method. Excess detection reagents may have been used or color allowed to develop for too long. Try to apply appropriate amounts of detection reagents and closely monitor the color development. Once major bands become visible, stop development immediately by rinsing the filter with distilled water several times.

8. Unexpected bands show up on the x-ray film or membrane filter. This problem is most likely caused by nonspecific binding between the probes and different DNA species. To solve or prevent such a problem from occurring, increase the blocking time for the blotted membrane and elevate the stringency conditions for the hybridization and washing processes.

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