Southern Blot Protocol with Digoxigenin (DIG) probe JAX Mice strain: 023099 C57BL/6J Tg(C9orf72_i3)112Lutzy/J Tail Genomic DNA extraction with QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, cat. no.69504 or 69506) 1. Sample 0.5 cm of tail. NOTE: genomic DNA extraction works better on tail tips from mice <4 weeks of age. 2. Transfer to a 1.5 ml Eppendorf tube. Add 180 ul Buffer ATL. Add 20 ul Proteinase K, mix by vortexing, and spin down briefly. Make sure tail tip is submerged in solution. Incubate at 56 o C in a dry oven overnight. 3. The following day, add 200 ul Buffer AL. Mix thoroughly by pulse vortexing for 5 10 seconds. Incubate at 56 o C for 10 min. 4. Add 200 ul 100% EtOH. Mix thoroughly by pulse vortexing for 5 10 seconds. 5. Briefly spin down and transfer the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at 6000 x g (8000 rpm) for 1 min. Discard the flow through and collection tube. 6. Place the spin column in a new 2 ml collection tube. Add 500 ul Buffer AW1. Centrifuge for 1 min. at 6000 x g. Discard the flow through and collection tube. 7. Place the spin column in a new 2 ml collection tube. Add 500 ul Buffer AW2. Centrifuge for 3 min. at 20,000 x g (14,000 rpm). Discard the flow through and collection tube. 8. Transfer the spin column to a new 1.5 ml Eppendorf tube. 9. Elute the DNA by adding 100 ul of nuclease free water (ThermoFisher Scientific, cat. no. AM9939) to the center of the spin column membrane. Incubate for 1 min. at room temperature (15 25 o C). Centrifuge for 1 min. at 6000 x g. 10. Repeat step 9 by adding an additional 102 ul of nuclease free water onto the column and eluting into the same Eppendorf tube. 11. Measure DNA concentration of the combined elutions by NanoDrop (Thermo Scientific) or other spectrophotometer. Genomic DNA digestion Digest 200 ul of DNA with XbaI (New England Biolabs, cat. no. R0145L) in a final volume of 250 ul. Use 3 ul of enzyme at 20U/uL and incubate in an oven overnight at 37 o C. In the morning, add an additional 1 ul of enzyme and digest 3 more hours. Agarose electrophoresis 1. Make a gel: Prepare a 0.7% agarose gel with SeaKem LE Agarose (Lonza, cat. no. 50005) in 500 ml of 1X TAE Buffer. Use a large gel tray (21 x 25 cm) with a comb that has the appropriate numbers of wells. 2. Run the gel: Load samples (total volume of 40 ul for a comb of 36 wells), 5 ng DIG labeled DNA Molecular Weight Marker III (Sigma Aldrich, cat. no. 10528552001) and 10 ul GelPilot 1 kb ladder (Qiagen, cat. no. 239085). Run at 70 mv for 16 hours. Blotting 1. Prepare the bridge: use one gel tray as a stand and Whatman 3MM Chromatography paper (GE Healthcare Life Sciences, 8x10, cat. no. 3030 866) to wrap the gel tray. Place the gel tray, flat bottom up, in a large, flat container (Cescolite CL810T, 8x10 ) and fill with about 1.5 L of 10X SSC Buffer. 10X SSC Buffer: Dilute 20X SSC Buffer (Fisher Scientific, cat. no. BP132520) 1:2 with double filtered deionized (DI) water. 2. Do not treat the gel with HCl (omit depurination) 1
3. Denature the gel 2 times for 15 min. in 500 ml of Denaturing solution prepared fresh (do not reuse). Use a MaxQ 2000 orbital shaker (Thermo Scientific, cat. no. SHKE2000). Denaturing solution: 87.66 g NaCl, 20 g NaOH for 1 L. 4. Decant off the denaturation solution. 5. Rinse the gel by filling the container with tap DI water and decanting off the water. 6. Add approximately 500 ml of Neutralization solution. Return the gel to the shaker platform and gently shake for about 30 min. at room temperature. Decant off the neutralization solution. Neutralization solution: 87.6 g NaCl, 61 g Tris Base for 1 L, ph 7.4. 7. Mount Capillary blotting w/ Roche positively charged membrane (Sigma Aldrich, cat. no. 1141724000), use water then 0.2X SSC Buffer to pre wet the membrane. Gel should be inverted (loading wells facing down). Use 4 layers of Whatman 3MM Chromatography paper on top of the membrane, 8 layers of Grade GB005 Blotting Paper (GE Healthcare Life Sciences, cat. no. 10426994) and top with paper towels. Place a weight (no more than 500 g) on the blotting apparatus. Allow to transfer with 10X SSC Buffer overnight (typically 16 hours). The next day, replace wet paper towel with dry ones and transfer for a couple more hours. 8. After transfer is complete, remove all blotting paper leaving the membrane on top of the gel. Mark the right bottom corner to indicate lane 1 using a soft lead pencil or razor blade. Carefully lift the membrane from gel. Transfer the membrane to a gel tray and allow to air dry for several minutes. 9. Cross link DNA in a UV cross linker (Fisher Scientific, cat. no. 13 245 221) with DNA side facing up. Use Energy mode with 120000 μj. 10. Rinse the membrane in 0.2X SSC Buffer for 5 min. Wrap the damp membrane, supported by a sheet of Whatman paper, and store at 4 C if hybridization does not follow immediately. Pre hybridization 1. Prepare DIG Easy Hyb Buffer: Add 64 ml of carefully pre warmed nuclease free water in two portions to the plastic bottle of DIG Easy Hyb Granules (Sigma Aldrich, cat. no. 11796895001), dissolve by stirring immediately at 37 C. Dissolving normally takes 10 15 min. 2. Pre heat appropriate volume (pre hyb + hyb) of DIG Easy Hyb at 48 C in a hybrization oven (VWR/Boekel, cat. no. 230402V). Follow charts below for volumes if using a glass roller bottle (35x300 mm, VWR, cat. no. 502 0300R) for hybridization. For pre hybridization Membrane size (cm) 20X25 20X15 Volume of DIG Easy Hyb (ml) 50mL 30mL For hybridization Membrane size (cm) 20X25 20X15 Volume of DIG Easy Hyb (ml) 17.5mL 14mL 3. Following crosslink and rinse in 0.2X SSC Buffer (above), roll the damp membrane, insert into an incubation roller bottle, and then rinse the membrane with 10 ml of double filtered DI water to make it stick to the wall of the bottle. Avoid trapping bubbles between the membrane and the glass. If membrane overlaps itself in the bottle, top the membrane with a nylon mesh (VWR, cat. no. 230415V [10x15 cm] or 230423V [23x23 cm]) as a spacer. Decant water. 4. Incubate membrane in appropriate volume of Pre Hyb Solution at 48 o C for 3 4 hours with rolling. Set roller speed at 10. 2
Hybridization 1. Add DIG labeled oligonucleotide probe (GGGGCC) 5 (see details below) into pre warmed DIG Easy Hyb to make a 100ng/mL solution. Mix well by inverting. Do not vortex and avoid foaming. 2. Decant pre hybridization buffer. Immediately add probe/dig Easy Hyb mixture slowly onto the inner side of the roller bottle (avoiding creating bubbles). 3. Incubate with rolling speed of 10 (gentle agitation) at 48 C overnight. NOTE: Hybridization buffer can be collected and reused. Store at 20 o C. Stringency Washes 1. Pre warm washing buffers at 48 C and 68 C by place washing buffers in oven the night before. 2. Low stringency wash: a. Wash membrane in 100 ml of 2X SSC Buffer containing 0.5% SDS for 15 min. while the oven is being ramped from 48 C to 68 C. Increase the rolling speed to 20. b. Decant washing buffer, and wash again in fresh buffer at 68 C for 15 min. 3. High stringency wash: a. Wash for 15 min. in 100 ml of 0.5X SSC Buffer containing 0.5% SDS at 68 C. b. Wash again for 15 min. in 100 ml of 0.15x SSC containing 0.5% SDS at 68 C. Antibody detection Preparation of solutions for detection: Maleic Acid Buffer: 0.1 M Maleic acid (Sigma Aldrich, cat. no. M0375 500G), 0.15 M NaCl; adjust with NaOH 10 N solution to ph 7.5. Store at room temperature (15 25 C). Blocking Solution: 1. 10X Stock solution: Dissolve Blocking Reagent (Sigma Aldrich, cat. no. 11096176001) in 1X Maleic acid buffer to a final concentration of 10% (w/v) with stirring and heating either on a heating block or in a microwave oven. Autoclave the stock solution and store at 4 C. 2. 1X working solution: Dilute 10X Blocking Solution at 1:10 with 1X Maleic Acid Buffer. NOTE: Always prepare fresh working solution. Washing buffer: 0.1 M Maleic acid, 0.15 M NaCl; ph 7.5; 0.3% (v/v) Tween 20. Store at room temperature (15 25 C). Antibody Solution: Centrifuge Anti Digoxigenin AP antibody (Sigma Aldrich, cat. no. 11214667001) for 5 min. at 10,000 rpm in the original vial prior to each use, and pipet the necessary amount carefully from the surface. Dilute Anti Digoxigenin AP 1:10,000 (75 mu/ml final) in Blocking solution. Once thawed keep the antibody at 4 o C. Detection Buffer: 0.1 M Tris HCl, 0.1 M NaCl, ph 9.5 (at ~20 C). Store in plastic container at room temperature (15 25 C). NOTE: ph is critical. Detection procedure 1. After stringency washes (above), rinse membrane for 5 min. in Washing Buffer in an open tray (Cescolite CL810T, 8x10 ) on an orbital shaker at room temperature. Use enough buffer (~200 ml) to submerge the membrane. NOTE: Do not let the membrane dry during handling. 2. Transfer the membrane to a plastic container (Genhunter, cat. no. B111 or B112, 20.7x15.9) and add 100 ml Blocking Solution into the container. 3
3. Incubate with gentle rocking at room temperature for 30 min. to 2 hours on a rocker or the rocking platform of the hybridization oven (UVP, cat. no. 95 0030 01). NOTE: This blocking step can last up to 3 hours without affecting results. 4. Discard Blocking Solution. Add 100 ml Antibody Solution. 5. Incubate for 30 min. with gentle rocking at room temperature. 6. Discard the Antibody Solution. 7. Wash membrane 3 15 min. in 100 ml Washing Buffer on a shaker with a shaking speed of 150 rpm (vigorously). 8. Briefly rinse membrane with 20 ml Detection Buffer. Equilibrate membrane 5 min. in 100 ml fresh Detection Buffer. Chemilumiscence 1. Using gloves, add the chemiluminescent substrate to the blot, as follows: Cut a sheet protector (Universal Top loading sheet protector, clear) so it can open with only one side sealed. Place the membrane (DNA side facing up) on top of the inner side of a development plastic sheet. For every 100 cm 2 of membrane, apply 1 ml (20 30 drops) of Ready to use CSPD (Sigma Aldrich, cat. no. 11755633001) or CSPD Star, drop wise, over the surface of the blot. Apply the top plastic sheet onto the membrane and immediately roll slowly (and without too much pressure) with a 50 ml pipet from the sealed edge to the other side until the CSPD is spread evenly over the membrane. NOTE: Do not let air bubbles form between the membrane and the upper surface of the sheet protector. 2. Incubate for 5 min. at room temperature. 3. Squeeze excess liquid from the sealed side out of the membrane and seal the 2 edges of the protector that are adjacent to the 1 st sealed edge of the sheet protector. 4. Squeeze out bubbles and flatten surface from the sealed edges to the unsealed edge. Seal the last edge. 5. If you are using CSPD, incubate damp membrane (in the sealed container) for 10 min. at 37 C to enhance the luminescence reaction. Protect from light during incubation. 6. Expose the sealed envelope (containing the membrane) at room temperature to Bioluminescence X ray film (Sigma Aldrich, cat. no. Z370371) for 15 min. 7. Feed the film through the developer. If signal is too strong, expose a new film for 10 min; if signal is weak, expose for up to two hours. Adjust exposure further (5 min. 12 hours depending on the strength of the signal). The signal is stable for at least 48 hours. With a starting DNA of 1ug, a typical exposure takes up to 16 hours. 4
Digoxigenin (DIG) oligonucleotide probe: (GGGGCC) 5 Integrated DNA Technologies, Inc. 5