Tg 008 Southern Blotting

Transgenic Mouse and Gene Targeting FacilityTg 008 Southern Blotting

Materials and EquipmentHybaid hybridization oven (ISC BioExpress, H-9250)Vacuum oven (VWR, 52201-218)Ludlum Geiger CounterRadiation Shields and lock box (from various vendors)Mini-centrifuge (VWR, 20668-212)Thermolyne Dri-Bath, Type 17600 (VWR, 35756-006)Platform Shaker, New Brunswick Scientific (VWR, 40000-300)Electrophoresis Separation Systems (Owl)QIAquick Nucleotide Removal Kit (Qiagen, 28304)Prime-It II Random Primer Labeling Kit (Stratagene, 300385)[alpha-32P]dCTP (ICN)Salmon Sperm DNA (Stratagene, 201190)SDS solution, 20% w/v (Bio-Rad, 161-0418)Zeta-Probe Genomic Tested Blotting Membrane (nylon) (Bio-Rad, 162-0196)Gel Blot Paper GB002 (Schleicher %26amp; Schuell, 34550)Gel Blot Paper GB004 (Schleicher %26amp; Schuell, 34340)Whatman paper, 3MMContrad 70 detergent (Fisher, 04-355-1)8 x 10 film cassette (Fisher, FB-XC-810)14 x 17 film cassette (Fisher, FB-XC-14178 x 10 Kodak X-OMAT AR film (XAR) (Fisher, 05-728-41)14 x17 Kodak X-OMAT AR film (XAR) (Fisher, 05-728-42)8 x 10 and 14 x 17 intensifying screen (Fisher, FB-IS-810 and FB-IS-1417)ART pipette tips (Rainin)

ProtocolDNA Preparation/Transfer to Membrane1. Digest 10 μl (5μg) of the sample DNA with the appropriate restriction enzyme.Note: At least 15 units of restriction enzyme should be used. Therefore, the volumes of enzyme and water may need to be adjusted.Conditions:2. Mix by pipetting several times with pipette and digest overnight at the appropriate temperature for the enzyme used (Generally 37℃). This is necessary to ensure proper digestion of the genomic DNA. 3. After overnight digestion, quick spin the samples and pipet up and down a few times to check the viscosity. If the sample is still viscous, add 1 μl enzyme and digest for an additional 1-3 hours. (Reaction may be stopped at this point by adding 0.5 μl 0.5 M EDTA for the 25 μl volume. Refrigerate sample until ready to run gel. Heat sample for 10 min. 42-56℃ and immediately load on gel.) Run digested DNA sample on a 0.8% TAE (or TBE) agarose gel. Be sure to include both a negative and a positive control and a size marker. Run the gel at no greater than 60 volts until proper separation of the desired bands is achieved. This generally takes from 6-18 hours depending on the expected size differential between the bands to be analyzed. Take a picture of the gel. If size markers have been used, be sure to use a UV ruler so that the size of the bands can be estimated. 4. Trim the gel to remove the size markers since these will often hybridize strongly to with the probe. Also trim away excess areas of the gel which do not contain DNA of interest.

5. Depurinate the gel by gently shaking the gel in 0.2M HCl for 10 minutes. This step hydrolyzes the DNA, breaking it into smaller fragments which are transferred more quickly to the nylon membrane. However, care must be taken since over incubation will hydrolyze the DNA into very small fragments which will not bind efficiently to the membrane. Therefore, it is not advised to perform this step unless the bands of interest are expected to be greater than 10 kb in size. 6. Gently shake the gel in 0.5 M NaOH/1.5 M NaCl for 30 minutes. Cut two sheets of GB002 gel blot paper and one sheet of Zeta-Probe GT membrane to the exact size of the gel. Place the cut sheet of membrane into distilled water for 5 minutesbefore using.

7. Cut Whatman 3MM to cover the base in the Southern blot apparatus (see diagram above). Place enough transfer buffer in the transfer dish to immerse the Whatman paper (that is hanging over the base) to soak up the buffer. Before the gel is placed on the Whatman paper be sure the surface is completely soaked with buffer. After placing the gel on the Whatman paper, remove any bubbles from beneath the gel by pressing them away to the sides. 8. Carefully position the wet Zeta-Probe membrane on the gel and remove bubbles that may be between the gel and the membrane. 9. Moisten the GB002 sheets in buffer and position them on top of the Zeta-Probe membrane. Remove all bubbles. 10. Saran Wrap is placed across the base of the apparatus to cover the transfer buffer. Bring the Saran Wrap across the base to the point where the gel begins. This is to seal the apparatus and prevent evaporation of the transfer buffer. This also prevents buffer from “wicking” from the whatman paper to the membrane directly thus bypassing the gel and not effectively transferring the DNA. Position paper towels (about 15 cm high) or 15-20 sheets of GB004 on top of the GB002 sheets. Cover the paper towel stack with a glass or plastic plate. Place a weight on top of the stack to facilitate transfer. However, care should be taken not to add excessive weight, which will compress the gel, and retard the capillary transfer. Transfer overnight.11. After transfer, separate the membrane from the gel and rinse the membrane briefly in 2X SSC buffer. Place the filter between two sheets of Whatman paper and bake at 80℃ in the vacuum oven for one hour to crosslink the DNA to the membrane. The dried membrane is stable at room temperature. The membrane can be stored dry between two sheets of GB002 in a plastic bag at 23-25℃.

Preparation of Radiolabeled ProbeNote: Wear lab coat, radiation badge, gloves, and eye shield while working with 32-P.

1. Turn on hybridization oven and heat to 65℃. Warm prehybridization buffer in 65℃ water bath. Note that the prehybridization buffer tends to precipitate. Make sure that the buffer is completely resuspended before using.2. Survey outside and inside of oven, work area (including floor), and other equipment in room with Geiger counter and indicate result on log sheet. Any areas of Geiger activity must be cleaned up before proceeding.3. Roll membrane(s) between mesh into tube so that prehybridization buffer will coat all surfaces.4. Add warmed prehybridization buffer to tube and incubate at 65℃ for at least two hrs. For large tube use 25-50ml prehybridization buffer depending on how many blots are being hybridized. For small tube use 10-20ml prehybridization buffer.Note: Work behind an acrylic shield for all following procedures.5. Remove [32P] dCTP from freezer and place behind shield in biological cabinet.6. Prepare Initial Reaction Mixture as follows:7. Boil reaction in heating block for 5 min. Note: be sure block wells are filled with H2O.8. Centrifuge briefly to collect condensation.9. Add 10μl 5X prime dCTP buffer to reaction mix.

10. Add 5μl labeled nucleotide [32P] dCTP to mixture (check the specific activity).Mix contents thoroughly with tip.When working with radioisotope use aerosol resistant tips (ART) to avoid pipetman and other contamination.11. Add 1 μl Exo (-) Klenow Enzyme. Mix contents thoroughly with ART tip.12. Incubate reaction in the heating block at 37℃ for 10 min.13. Stop reaction by adding 2μl Stop Mix (part of Stratagene Kit).14. Nucleotide removal and DNA clean-up procedure as follows:Following manufacturer’s instructions, pipette probe onto Lauryl Sepharose column (from either Qiagen or Stratagene). Centrifuge and discard column in solid waste container marked for 32P.Liquid in tube is checked with Geiger counter for activity before proceeding. Add Salmon Sperm to mixture for a final conc. of 100μg Salmon SpermDNA/1 ml hybridization solution. Stock solution is 10 mg/ml.15. Make a hole with a syringe needle in top of tube to release excess pressure and boil the probe for 10 minutes in the heating block must be kept behind acrylic shield. Syringe needle will be stored/discarded in capped centrifuge tube behind acrylic shield and discarded in solid waste.16. While probe is boiling, place 32P in acrylic lock box and return box and labeling kit reagents to freezer.

Hybridization and Exposure to Film1. Remove tube(s) from hybridization oven. Once the probe has boiled, quick spin it, and add immediately to the prehybridization buffer in the tube. Do not pipette the probe down the side of the tube, as this will cause the probe to be distributed unevenly across the membrane. down side of tube. Alternatively, the probe can also be placed on ice until mixture is added to prehybridization solution. Note: remember to perform all these procedures behind an acrylic shield. 2. Clean and monitor radioisotope bench area. Record date and amount of isotope used in logbook. Store all solid waste in a secured bag, labeled and placed in the solid waste container. 3. Hybridize overnight in 65℃ oven. (Before leaving room, check caps on tubes for any leakage.)4. After overnight incubation, remove hybridization solution by pouring solution into the 32P liquid radioactive waste container. A funnel placed on top of the container allows for pouring the hybridization solution without spills. Note: With the following washes, the membrane is kept in the tube it was hybridized in and all washes are performed in the hybridization oven. 5. Wash membrane(s) 1x for 15 min. in 2X SSC + 1% SDS, RT. Discard wash in 32P liquid waste container. 6. Wash membrane(s) 2x for 30 min. each in 0.1% SDS, 65℃ . Discard first wash in liquid waste container. If %26lt;1 mR/h the second wash may be disposed of in the designated lab sink. 7. After last wash, remove membrane(s) and dry the membrane(s) briefly between two sheets of Whatman paper. (Discard Whatman paper in 32P solid waste container.) Wrap membrane(s) in saran wrap and place in film cassette. Expose to X-ray film overnight in -80℃ freezer. 8. Soak tube(s) and mesh separators in Count-Off solution overnight. Then rinse with dH2O and hang to dry. Used Count-Off may be disposed of in the sink.9. Monitor oven and bench area with Geiger counter. Record results and amount of 32P waste in logbook. Once a week perform wipe tests on all surfaces with possible exposure to radioisotope. 10. Remove film cassette from the �80℃ freezer. Develop immediately or let cassette warm to room temperature before developing. Follow instructions on developer for developing film. If exposure needs to be for a longer time, ｒｅｐｌａｃｅ with new X-ray film and again place cassette in �80℃ freezer for extended period of time. Develop when needed. 11. After developing film wrap membrane(s) in foil, label, and store in acrylic box for future use. Solutions and Buffers Transfer Buffer100 μg salmon sperm DNA/ml (added with the radioactive probe)Note: Store hybridization buffer at 4oC and warm to 65oC prior to use since dextran sulfate precipitates during storage and will need to be resuspended completely to achieve optimal results.

20X SSC Stock SolutionAdd 175.3g NaCl and 88.2g sodium citrate to 800 ml distilled water and pH to 7.0. Add water to 1000 ml and autoclave to sterilize.

Removing Probe From Nylon Membranes1. If reprobing is desired, do not allow the Zeta-Probe membrane to dry between hybridizations. The membrane should be stripped as soon as possible after autoradiography.2. Wash the membrane twice for 20 minutes each in a large volume of 0.1X SSC/0.5% SDS at 95℃.3. Sandwich the damp membrane between two sheets of Saran Wrap, and apply it to X-ray film to check that the entire amount of probe has been removed.4. At this point the membrane may either be rehybridized or dried and stored at room temperature until needed.