Appendix 5 Ricardo V. Lloyd and Arie Perry

FISH APPENDICES FISH on Paraffin Protocol I. Deparaffinize/Pretreat CitriSolv-10 min x2 Isopropynol-3 min x3 Air dry 0.2N HCL-20 min Running H2O-5 min dH2O-3 min II. Target Retrieval Steam cook in citrate buffer pH 6.0-20 min Slow cool at room temperature while in buffer-20 min Running H2O-5 min dH2O-3-5 min III. Tissue Digestion Pepsin digestion at 37 °C-30 min (Pepsin at 4mg/ml=160mg pepsin + 40ml dH2O) Running H2O- 5 min 2X SSC wash-5 min IV. Codenaturation/Hybridization Air dry slide Dilute (red + green) probe in DenHyb (1/10-1/200) or other hybridization solution Apply probe or probe cocktail to target tissue, 10μl for half a slide, 20μl for full slide Coverslip and gently press out air bubbles Codenature on slide tray to 90 °C-13 min Hybridize in humidified chamber at 37 °C overnight V. Post-Hybridization Wash Place slides in 50% formamide/1X SSC (20 ml formamide + 20 ml 2X SSC) until coverslips fall off Wash in 50% formamide/1X SSC at RT- 3-5 min Wash in 2X SCC at RT –2 min x2 VI. Viewing Air dry slide Counterstain with 10μl DAPI (1.0μg/ml) Coverslip and gently press out excess DAPI View under fluorescence microscope Growth of Cloning Vector Bacteria LB Agar (for plates) - -------8 tablets----400ml H2O 2xYT Broth (1L) 16g Tryptone 10g Yeast extract 5g NaCl dH2O Autoclave both solutions for 15 min. Cool down to approximately 50° C. Add appropriate antibiotics to LB Agar & 2xYT broth Pour LB Agar in the plates (100/plate). Cool plates at RT. With a sterile inoculating needle streak the bacteria (BAC) into the surface of a LB agar plate. Grow bacteria in 37 ° C incubator overnight. Isolation of Cloning Vector DNA (e.g. Qiagen Midiprep Plasmid Kit) Pick a single colony from a freshly streaked plate and inoculate a starter culture of 5ml LB broth containing appropriate antibiotics. Incubate for 8 hrs at 37 °C with vigorous shaking - ---250-300 rpm. Liquid should appear cloudy. Inoculate 1.5 ml of starter culture into 200ml of LB broth in a 1000ml Erlenmeyer flask. Incubate overnight at 37° C in a rotary shaker at 250-300 rpm. The culture should appear cloudy. Transfer culture into four 50ml polypropylene bottle and spin at 6250 rpm for 20min. at 4 °C on Sorvoll RC superspeed. Remove all supernatant. Resuspend the bacterial pallet in 10ml buffer P1 (see solutions). Add 10 ml buffer P2 (see solutions) to each tube. Mix thoroughly and gently by inverting 4-6 times and incubate at room temperature for 5 min (do not vortex). Should appear viscous. (Check buffer P2 before use for SDS precipitation due to low storage temp. If necessary dissolve SDS solution by warming to 37 ° C.) Add 10ml chilled buffer P3 (see solution), mix gently by inversion, and incubate on ice for 15-min. fluffy white materials forms. Centrifuge at 13000 rpm (Sorvall) for 30 min at 4° C. Remove supernatant containing plasmid DNA promptly. Re-centrifuge supernatant at 13000 rpm for 15 min. at 4° C. Remove supernatant containing plasmid DNA promptly. Precipitate the DNA by adding 0.7 volume of RT Isopropanol to lysate. Centrifuge at 11,000 rpm for 30 min. at 4° C. Redissolve the DNA pallet in 500ul of TE –pH-7.0. Add 4.5ml of QBT buffer. Equilibrate a Qiagentip 100 by applying 4ml QBT buffer and allow the column to empty by gravity flow. Apply the DNA solution from step 10 to Qiagen tip and allow it to enter the resin by gravity flow. Wash Qiagen tip with 2x10 ml QC buffer Elute DNA with 5x 1ml QF buffer (pre warmed to 65° C). Collect in Oak Ridge 10ml centrifuge tube. Precipitate DNA by adding 3.5ml RT Isopropanol to the eluted DNA. Mix and centrifuge immediately. At 11000 rpm for 30 min. at 4 °C. Carefully decant the supernatant without disturbing the pellet. Wash DNA pellet with 2ml of RT. 70% ethanol and centrifuge at 11000 rpm for 10 min. Carefully decant the supernatant without disturbing the pellet. Airdry pellet for 10 min. Re-dissolve DNA in 25 ul of TE—pH-8.0. Take OD reading in spectrophotometer. Qiagen Solutions 1. Solution P1 500ml (Ref). Tris base 3.03g EDTA(disodium dihydrate) 1.86g dH2O 400ml Adjust pH to 8.0 w/HCl Adjust final volume to 500 ml Add 50 mg Rnase A (0.5ml of 100mg/ml solution) 2. Solution P2 500ml (RT) NaOH (pellet) 4g 20% SDS solution 25ml dH2O 475ml 3. Solution P3 500ml (Ref) Potasium Acetate 147.25g dH2O 250ml Adjust pH to 5.5 w/Glacial Acetic Acid (approx. 55ml) Adjust final volume to 500 ml. Labeling of Fish Probes Mix reagents on ice. In each tube add the appropriate reagents in the following order. Procedure can be scaled up in a linear fashion for up to 3μg DNA. Distilled water 45 (17+X) l (Gibco Kit #18247-015) 10x dNTP Mix 5μl (Home made) Labeled dUTP 2μl (Roche-# 137322 Fluorescein-12-dUTP; #1534378 Rhodamine 5dUTP) 1 g DNA Xμl (concentration determined by spectrophotometer) 10x Enzyme mix (containing DNase1 and DNA polymerase 1) 5μl (Gibco #18010-017) Polymerase 1 5μl (Roche #642720) Total Volume ---------------------   45μl Incubation: Quickly vortex the above mixture and place it for 120 min. at 15°C in PCR Thermocycler or in Isotemp Refrigerated Circulator. Stop reaction by adding 5m l Stop buffer(Gibco kit#18247-015), vortex and keep it in -20°C until next step. Probe Precipitation:   1x 3x Add to the labeled DNA probe sample 50μl 150μl Human Cot1-DNA(1 m g/m l)(Gibco#15279-011) 50μl 150μl Salmon Sperm DNA (5mg/ml)(Sigma D-7656) 10μl 30μl 0.1 Vol. NAACO (3M/pH=5.2)(Sigma S-7899) 11μl 33μl 2 Vol. 100% Ethanol (- 20 °C) 242μl 726μl Mix well and incubate 60 min. at -20°C. Centrifuge 30 mins/4°C/15000 rpm. Discard supernatant and dry precipitate for 2-3 mins/4°C in Speed Vac. Concentrator. Do not over dry. Dissolve precipitate in 10μl distilled water/37°C waterbath overnight. Store in -20°C.