DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols^©

Southern blot hybridization

(last modified January 26, 2000)

Protocol

for the labeling (Megaprime, Amersham) of a DNA probe, the (pre-)hybridization of a Southren blot and its washing, exposure and stripping (kindly provided by Bert Bakker and Els Voorhoeve)

Target group

The technicians who are working in the clinical DNA-diagnostic department

Responsibilities

The technicians using the megaprime DNA labeling and hybridisation for DNA-diagnostic analysis of specific diseases are responsible to perform these procedures according to this protocol.

Definitions

(M): available at the storehouse of the Sylvius Laboratory
RT: room temperature
min: minutes
sec: seconds
cps: counts per second

Description

In this protocol is described how DNA from a variety of sources can be labeled in vitro to high activity with [alpha-³²P]-dCTP. Random sequence nonameres are used to prime DNA synthesis on denatured template DNA at numerous sites along its length. The primer-template complex is a substrate for the "Klenow" fragment of DNA polymerase I. By substituting a radiolabeled nucleotide for a non-radioactive equivalent in the reaction mixture, newly synthesized DNA is radioactively labeled. The absence of the 5'-3' exonuclease activity associated with DNA polymerase I ensures that labeled nucleotides incorporated by the polymerase are not subsequently removed as mono-phosphates. The probe DNA is separated from the unincorporated nucleotides using a Sephadex G50-column. The stopmix used, consists of two dyes (blue and red). The probe with the incorporated label migrates with the blue fraction (high molecular weight) which runs faster than the red fraction (low molecular weight), containing the unincorporated nucleotides.

The radioactive labeled probe hybridizes with those restriction fragments, immobilized on a nylon filter (see Southern blotting - ALG004), that contain sequences complementary to the probe. To block the nonspecific attachment of the probe to the surface of the filter, denatured and fragmented herring sperm DNA can be used (optional). This blocking agent is added to the hybridisation mixture prior to pre-hybridisation. The polyethyleneglycol (PEG) used in the hybridisation mixture is to increase the effective probe concentration and enhances the signal needed for single copy detection on genomic Southern blots. The DNA is excluded from the volume, which is occupied by the PEG. Washing of the hybridised filters is performed with increasing stringency of the wash solution which is obtained by decreasing the salt concentration of the washing solution. Finally, an autoradiographic image of the filter is obtained by exposing an X-ray film to the filter or using the Phosphor Imager.

Durables

-80^oC Ultrafreezer, Denley, Beun de Ronde B.V.
Appligene TFX-35M U.V. Bench, B & L systems
cassette; 20 x 40 cm Auto-G (1190 1050, Konica) or 35 x 43 cm Auto-G (1190 1090, Konica)
Elga Ultra Pure Water System, Salm & Kipp
heat block, Unitek HB130, Biozym
hybridisation trays, small: 15 x 8 cm, large: 23x8 cm, Blokker
intensifying screen; 35 x 43 cm Blue Regular (1180 2190, Konica) or 20 x 40 cm Blue Regular (1180 2150, Konica)
microcentrifuge, type 5414, Eppendorf, Merck
microwave, Amana RS591SS, Salm & Kipp
Phosphor Imager, B & L Systems
pipetman micropipetten, P20, P200, P1000, Gilson (M)
shaking waterbath, type 1083, GFL, Salm & Kipp
trays, for stripping filters, 20 x 20 cm, Blokker
waterbath, GFL, Salm & Kipp

NOTE: equivalent durables may be used

Consumables

blue tips, Greiner (M)
Lithium Power Cells, Ultralife Batteries, (M)
nylon-wool
paper towels (M)
Pasteur Capillary Pipettes, long size (230 mm) and short size (150 mm), WU Mainz (M)
plastic waste bag (M)
Safe-Lock tubes, 1.5 ml, Eppendorf (M)
Saran Wrap (M)
tips C10, 170558, Biozym
tissues (M)
underlayment (M)
X-Ray films, X-OMAT^TM AR, 35 x 43 cm and 20.3 x 25.4 cm Kodak (M)
yellow tips, Greiner (M)

NOTE: equivalent consumables may be used

Chemicals

blue dextran (D-5751, Sigma)
bromophenolblue (B7021, Sigma)
developer, Roentogen, 1220 1030, Konica
ethylene diamine tetra acetate (EDTA), Baker (M)
phosphoric acid (H₃PO₄), Baker (M)
Megaprime DNA labeling system (RPN 1607, Amersham)
sodium monohydrogen phosphate (Na₂HPO₄.2H₂O), Baker (M)
poly ethylene glycol (PEG) 6000, USB (M)
phenol red (phenolsulfonphtalein) (P-4758, Sigma)
sodium dodecyl sulphate (SDS), USB (M)
Sephadex G50, 17-0573-02, Pharmacia Biotech
water, autoclaved Elga water (see WFW01)
X-Ray Fixing Solution, Tetanal Roentogen Superfix (1220 1045, Konica)

NOTE: equivalent chemicals may be used

Solutions

For the solutions marked with a * a special form (with the protocol on it) is filled out. The other solutions are ready to use or made as described here.

SDS; 10% ultra PURE (15553-019, Life Technologies)
*2 x stopmix (see WFS06); 25 mM EDTA, 2.0% Blue Dextran, 0.2% Phenol Red
SSC; 20 x stock solution 3.0 M NaCl, 0.3 M Sodium citrate, pH7.0 (15557-028, Life Technologies)
Dekasol; (808026, ICN Biomedical Inc.)
developer solution; for 1 l dilute 220 ml developer to 1 l with Elga water
NOTE: store the solution at room temperature. When this solution gets exhausted, discard it in the chemical waste deposit in the dark room (black label)
EDTA; 0.5 M pH8.0 (15575-012, Life Technologies)
fixation solution; for 2 l, dilute 400 ml X-Ray fixing solution to 2 l with Elga water (use a magnetic stirrer to get a homogeneous solution
NOTE: store in a 2.5 l bottle. When this solution gets exhausted discard it in the chemical waste deposit in the dark room (black label)
herring sperm DNA; 10 mg/ml (14430-011, Life Technologies)
*hyb-mix (NaPi/SDS/PEG, see WFH01); 125 mM Na₂HPO₄.2H₂O, 7 % SDS, 10 % PEG-6000, 1 mm EDTA
redivue alpha-³²P dCTP; 370 MBq/ml, 10 µCi/µl, 1 mCi, Amersham
Sephadex G50-solution; for 500 ml, dissolve 35-40 g of Sephadex-G50 in 500 ml TE-4, autoclave the solution and store at RT for a maximum of 1 year
strip buffer; 0.1 x SSC/0.5% SDS. For 2 l, dilute 10 ml 20 x SSC to 1,900 ml by adding Elga water in a 2 l cylinder and add 100 ml 10% SDS
*TE-4 (see WFT02); 0.01 M Tris-HCl, 0.1 mm EDTA, pH7.5
Tris.HCl; 1 M, pH7.5 (15567-019, Life Technologies)
washbuffer;
1. 2 x SSC/0.1% SDS
  NOTE: for 2 l, in a 2 l cylinder dilute 200 ml 20 x SSC to 1.78 l by adding Elga water and add 20 ml 10% SDS
2. 1 x SSC/0.1% SDS
  NOTE: for 2 l, in a 2 l cylinder dilute 100 ml 20 x SSC to 1.88 l by adding Elga water and add 20 ml 10% SDS
3. 0.3 x SSC/0.1% SDS
  NOTE: for 2 l, in a 2 l cylinder dilute 30 ml 20 x SSC to 1.95 l by adding Elga water and add 20 ml 10% SDS
4. 0.1 x SSC/0.1% SDS
  NOTE: for 2 l, in a 2 l cylinder dilute 10 ml 20 x SSC to 1.97 l by adding Elga water and add 20 ml 10% SDS
lambda-DNA; (27-4111-01, Pharmacia)
radio-active labeled lambda-DNA, see WFL02

DNA hybridization protocol

The probe labeling is started in the post PCR lab. All handlings involving [alpha-³²P]- dCTP are performed in the C-lab, according to the general C-Lab safety rules (see LP001). Always wear a lab-coat, gloves and safety glasses and protect yourself and others from radiation using the radiation protection shields present in the C-Lab.

Before starting the megaprime DNA labeling a waterbath is reserved (in the C-lab) and pre-warmed at 65^oC. Write down the date, your name and the number of small and/or big hybridisation trays you will use. Fill the waterbath with Elga water to approximately 1 cm above the platform. The hyb-mix is pre-warmed at 65^oC in the incubator in the post PCR lab.

The [alpha-³²P]- dCTP is stored at room temperature in the Redivue Isotope Dispenser.

PRE-HYBRIDIZATION
1. check if the waterbath is at 65^oC
2. thoroughly clean the hybridisation trays (first with "Jif", then with demi water) and dry them with paper towels. Wrap the trays used as a cover with Saran Wrap to prevent contamination
3. cut the filters to the appropriate size: maximal 14 x 7 cm for the small trays and 22 x 7 cm for the large trays
4. pour the appropriate amount of pre-warmed hyb-mix (65^oC) in the cleaned trays
  NOTE: pre-hybridisation is carried out at 65^oC
  - small tray; 25 ml hyb-mix for one filter and 40 ml for a maximum of 6 filters
  - big tray; 40 ml hyb-mix is used for one filter and 60 ml for a maximum of 6 filters
5. add 100 µl herring sperm DNA solution for each 10 ml of hyb-mix in the tray
  NOTE: this optional step is to block the non-specific attachment of the probe to the surface of the filter
6. mix gently and place the filters, DNA side up, in the hyb-mix. After adding the filters, mix gently to completely cover the filters with hyb-mix
7. place the cover on top of the tray and incubate in the shaking waterbath at 65^oC for 10-60 min
PROBE LABELING
The Megaprime^TM DNA labeling system from Amersham, stored in the post PCR freezer, is used for the labeling.
1. mark a 1.5 ml tube with the name of the probe which will be labeled
2. pipet 10.5 µl autoclaved Elga water at the bottom of the tube, add 2 µl primer mix (Mega prime kit, black capped tube) and 1 µl of a 10-25 ng/µl probe stock
  NOTE: if a more diluted probe DNA stock is used, the endvolume is kept at 11.5 µl by adjusting the water volume
3. denature this mixture for 5 min in the Heatblock at 100^oC
4. leave the tubes at RT for 5 min ( DO NOT COOL ON ICE !!)
5. spin down for a few seconds to collect the probe mix at the bottom of the tube
6. add 4 µl buffer/dNTP's (Mega prime kit, blue capped tube) and 1 µl Klenow polymerase (1 U/µl) (Mega prime kit, red capped tube)
7. mix the solution gently by tapping the tube; centrifuge shortly if necessary
8. keep the solution on ice until the label is added
  
  The following steps are performed in the C-lab
9. check the work tray for radioactive contamination; if necessary, remove and clean this contamination according to the guidelines and general rules for the C-lab
10. install a radioactive waste container in the tray
11. add 1.5 µl [alpha-³²P]-dCTP to the prepared reaction mix (it is not necessary to mix the solution afterwards)
12. carefully remove the contaminated tip and put it in the radioactive waste container
13. close the tube with the labeling mix and move the tube from the work tray to the 37^oC waterbath
14. cover the tube with the perspex cover to prevent unnecessary radiation
15. incubate for 10-15 min in the 37^oC waterbath
  
  Meanwhile prepare a Sephadex G50 column in a Pasteur pipet in the post PCR lab
16. insert a nylon-wool plug in a short Pasteur pipet just above the capillair using a long Pasteur pipet
17. place the short Pasteur pipet in a 1.5 ml tube in a rack
18. mark a second tube with the name of the probe used for labeling and place it in the rack opposite of the Pasteur pipet
19. fill the pipet with the Sephadex solution using a long Pasteur pipet
  NOTE: homogenize the Sephadex solution before use, prevent trapping of air bubbles in the column (remove air bubbles using the long Pasteur pipet)
20. add additional Sephadex until the packed column fills the pipet from the plug of glass wool to just above the constriction, ± 1 cm from the top of the pipet
21. remove the eluted TE-4 from the tube
  
  The following steps are performed in the C-lab
22. take the Sephadex columns and place them in a rack in the work tray
23. take the tube with the probe mixture out of the 37^oC waterbath and place it in the work tray
24. add 20 µl of 2 x stop-mix and transfer all on top of the Sephadex column, without changing tips (put the contaminated tip in the radioactive waste)
25. add 2-3 drops of TE-4 buffer with a Pasteur pipet at the top of the column so that all the radioactivity will flow into the column. Keep filling the column with TE-4 until elution of the blue fraction is complete
26. collect the blue fraction in the marked tube by transferring the column to this tube
27. upon complete elution of the blue fraction, return the column to the other tube and discard the column to the radioactive waste after complete TE-4 elution
28. test the incorporation of the label by keeping the tube with the blue fraction in front of the Minimonitor Geigercounter
  NOTE: for a good labeling, the pointer should go off the scale. If a radiation of less then 500 cps is measured, repeat the labeling
29. denature the double stranded probe by incubating for 5 min at 100^oC in the heat block (move the probe from work tray to heatblock in a small perspex container)
  NOTE: when a lambda-marker is needed, defreeze and denature it.
30. cool the probe on ice for 5-30 min
HYBRIDIZATION
1. to prevent the hyb-mix from cooling down too much, take one tray at a time from the waterbath. Remove all the filters from the tray, and put them on the turned cover of the hybridisation tray. Leave excess of the hyb-mix coming from the filter in the hybridisation tray
2. using a P200, add all of the probe, approximately 500 µl, (and optional 5-15 µl of the lambda-marker) to the hyb-mix and mix gently to get a homogeneous solution
3. place the filters one by one back in the hyb-mix, DNA side up
  NOTE: after addition of a filter, mix gently to cover the filter with hyb-mix (prevent trapping of air bubbles between/under the filters; remove air bubbles by replacing the filter)
4. incubate overnight at 65^oC in the waterbath under gentle shaking (to prevent the filters from adhering to one another)
  NOTE: prevent the tray from floating in the shaking waterbath
5. discard all of the produced radioactive waste in the appropriate waste container. Check the work tray for contamination and remove/clean if necessary. Fill out the booklet on top of the protection cover with your name, the date, time of usage and the isotope used
WASHING HYBRIDIZED FILTERS
The following steps are performed in the C-lab.
1. at least one hour before starting the washing procedure, put the wash solution in the 65^oC incubator in the post PCR lab
2. check the work tray for radioactive contamination. Remove and clean if necessary according to the guidelines and general rules for the C-lab (LP001)
3. install radioactive waste containers in the work tray, one for solid waste and one for liquid waste (preferably a glass container)
4. put Saran Wrap in the tray to discard the wet and possibly contaminated Saran Wrap from the cover of the hybridisation tray
5. remove the cover from the hybridisation tray and put the wet Saran Wrap on the clean sheet of Saran Wrap
6. remove the radioactive hyb-mix by pouring it in a glass container
  NOTE: the filters will stick to the bottom of the tray. Clean the side of the tray with a tissue to prevent contamination of the outside of the tray. DO NOT ALLOW FILTERS TO DRY AT ANY STAGE DURING THE WASHING PROCEDURE
7. add a little bit (20-40 ml) of wash solution I to rinse the filters and mix gently to remove all excess of radioactivity
8. replace with wash buffer I (50-100 ml for a small hybridisation tray) and wash at 65^oC for 15-20 min under gentle shaking in a waterbath
  NOTE: it is not necessary to cover the trays during the washing procedure. Check that the filters do not stick together
9. discard liquid radioactive waste above 10 cps in the liquid waste container
  NOTE: radioactive waste below 10 cps can be discarded in the sink
10. refresh wash buffer I and wash again for 15-20 min at 65^oC in a shaking waterbath
11. continue the washing procedure with wash buffer II and wash for 30-45 min at 65^oC in the shaking waterbath
12. remove filters one by one from the solution and measure the amount of radioactivity on the filter by keeping them in front of a Geiger counter
  NOTE: the filters have to radiate less than 10 cps. If more then 5-10 cps per filter are measured, continue washing with washbuffer III and if necessary also washbuffer IV, 30-45 min each at 65^oC in a shaking waterbath
13. replace the last wash buffer with 2 x SSC at room temperature
14. discard all of the produced radioactive waste in the appropriate waste container. Check the work tray for contamination and remove/clean if necessary. Fill out the booklet on top of the protection cover with your name, the date and time of usage of the work tray and the isotope used
15. take the trays to the post PCR lab for further handlings
16. remove most of the liquid from the filters by placing them on filterpaper
17. place the damp filter on a sheet of Saran Wrap
18. cover the filter with a second sheet of Saran Wrap in order to keep the filters moist. Expose the filters to X-ray film under safelight illumination in the dark room in a light-tight cassette with intensifying screen. Label the cassette with your name and the date

NOTE: the exposure time should be determined empirically but can vary from overnight to one week exposure (always at -80^oC in the freezer next to the elevator). The amount of radioactivity measured after the wash procedure determines which films are to be used. The sensitive Kodak X-OMAT^TM AR films are used for weak signals (1-3 cps per filter) and the Kodak Biomax MR or Konica AX films for strong signals (3-10 cps per filter). The exposure can also be performed using the PhosphorImager, which is about 10 times more sensitive

DEVELOPING EXPOSED X-RAY FILMS
Developing of exposed X-ray films is performed in the dark room wearing a disposable labcoat and gloves. Lock the door before starting the develop procedure.
1. remove cassettes from the -80^oC and defrost
2. pour the developer- and fixation solution in the appropriate tray
  NOTE: if necessary test the developer solution by placing a small piece of exposed film in the solution (the film should turn black within 30 sec). To test the fixation solution, a small piece of film is placed in the solution (the film should become clear within one minute). If the solutions do not meet these criteria, they should be discarded in the appropriate waste deposit (the tanks with the black label and a label "fixation" or "developer"
  NEVER DISCARD DEVELOPER- AND FIXATION SOLUTION IN THE SAME TANK; THIS WILL START A CHEMICAL REACTION (production of NH₃)
3. turn off the light and put the safety "red-light" on
4. incubate the X-ray film for 20 sec to 5 min in the developer solution under gentle shaking; check the amount of signal on the film by keeping the film in front of the safety light
5. wash the film briefly in water to remove most of the developer solution
6. incubate in fixation solution under gentle shaking until the film is completely clear
7. turn on the light, put the film in the tray with water, rinse with demi water and place the film in the incubator
8. turn on the incubator and leave the film to dry for 10-30 min
9. describe the film as follows;
  - complete DNA numbers above the lane
  - date of the blot (WBLOT)
  - restriction enzyme used in digestion procedure (ALG 004)
  - number of the blotgel (if more then one gel was blotted)
  - name of hybridised probe
  - all marker lanes
  - date of hybridisation
  - exposure time
10. store the films in the folder (for each month and each disease use a separate “showmap”)
STRIPPING OF HYBRIDISED FILTERS
1. Heat 0.5 l strip-mix in a 1 l erlenmeyer in the microwave oven until boiling
  NOTE: prevent the solution from boiling over by keeping the energy level of the microwave oven below 5
2. unwrap the hybridised filters from the Saran Wrap, put them in a clean tray (used for stripping of filters) and prevent the filters from becoming dry by adding a small volume (50-100 ml) of 2 x SSC
  NOTE: clean the cover of the tray
3. when the strip-mix reaches boiling temperature, remove the filters from the tray and leave them on the cleaned cover
4. remove the 2 x SSC and install the tray in the 65^oC shaking waterbath
5. pour the boiling strip-mix in the tray, immediately add all filters and incubate for 30 min in a 65^oC waterbath under gentle shaking
6. put the stripped filters neatly on filterpaper and dry them
7. stripped filters are stored, using the date present on the filter (date WBLOT), central in a drawer in the post-lab. For this, per month an envelope is labeled with month and year. Filters can also be used in further hybridisations procedures.

References

[1] Megaprime^TM DNA labelling systems, RPN 1604/5/6/7, Amersham LIFE SCIENCE

[2] Maniatis, Sambrook and Fritsch: Molecular Cloning part, 2^nd edition chapter 9.31 - 9.62