1.0 1249998432252 - iLAP - PROTOCOL_ARCHIVE 3D combinatorial DNA-MFISH of BAC1-BAC7 - preconfluent CSK Fix_2008-10-24 Simultaneous 3D-nuclear localization of 7 different genes by means of a combinatorial M-FISH. 1 1224852992152 Demo User SPLIT_STEP Cell preparation / Probe preparation cells and FISH probes are prepared for hybridization in parallel STEP_GROUP DNA FISH probe preparation FISH probe preparation: precipitate probe DNA together with human Cot1 and salmon sperm DNA and dissolve in hybridization buffer SINGLE_STEP prepare DNA mix for EtOH precipitation Mix the following: - ~100 ng nick-translated BAC-probe-DNA for each target gene - 0.7 x 2/3 x n µl salmon sperm DNA 10 mg/ml (n=number of total probes used for combinatorial labeling. e.g. 7 targets n = 12) - 5 x 2/3 x n µl cot1 DNA 1 mg/ml (n=number of total probes used for combinatorial labeling. e.g. 7 targets n = 12) - 11.3 x 2/3 x n µl ddH20 (n=number of total probes used for combinatorial labeling. e.g. 7 targets n = 12) - 2 x Vol. abs. EtOH - 1/10 x Vol. 3M sodium acetate pH 5.2 nick-translated DNA 10046 NUMERIC OPTIONAL amount of nick-translated DNA per target gene ng 100.0 100 salmon sperm DNA 10047 NUMERIC OPTIONAL amount of salmon sperm DNA 10 mg/ml: 0.7 x 2/3 x n µl n=number of total probes used for combinatorial labeling. e.g. 7 targets n = 12 µl 5.6 5.6 human cot1 DNA 10048 NUMERIC OPTIONAL amount of human cot1 DNA 1 mg/ml 5 x 2/3 x n µl n=number of total probes used for combinatorial labeling. e.g. 7 targets n = 12 µl 40.0 40 H2O 10049 NUMERIC OPTIONAL volume of H2O 11.3 x 2/3 x n n=number of total probes used for combinatorial labeling. e.g. 7 targets n = 12 µl 90.4 90.4 sodium acteate 10050 NUMERIC OPTIONAL volume of sodium acteate 1/10 x Vol. µl 17.0 17 abs. EtOH 10051 NUMERIC OPTIONAL volume of abs. EtOH 2 x Vol. µl 340.0 340 SINGLE_STEP put the DNA mixture at -70°C put the DNA mixture for 30 min. at -70°C temperature 10052 NUMERIC OPTIONAL temperature °C -70.0 -70 time 10053 NUMERIC OPTIONAL time minutes 30.0 30 SINGLE_STEP spin down in a centrifuge spin the precipitated DNA down in a centrifuge centrifugation speed 10054 NUMERIC OPTIONAL centrifugation speed rpm 13000.0 13000 centrifugation temperature 10055 ENUMERATION OPTIONAL centrifugation temperature °C 4 4 25 4 centrifugation time 10056 NUMERIC OPTIONAL centrifugation time minutes 15.0 15 SINGLE_STEP remove supernantant remove supernantant by pipetting carefully to not disturb the pellet. SINGLE_STEP rinse with 70% EtOH rinse pellet with 70% EtOH to remove salts: - add 80 µl 70% EtOH - centrifuge for 5 minutes SINGLE_STEP spin down in a centrifuge spin the precipitated DNA down in a centrifuge centrifugation speed 10057 NUMERIC OPTIONAL centrifugation speed rpm 13000.0 13000 centrifugation temperature 10058 ENUMERATION OPTIONAL centrifugation temperature °C 4 4 25 4 centrifugation time 10059 NUMERIC OPTIONAL centrifugation time minutes 5.0 5 SINGLE_STEP remove supernantant remove supernantant by pipetting carefully to not disturb the pellet. SINGLE_STEP dry pellet dry pellet at room temperature temperature 10060 NUMERIC OPTIONAL drying temperature °C 25.0 25 time 10061 NUMERIC OPTIONAL drying time minutes 20.0 20 SINGLE_STEP add formamide add formamide to a final concentration of 50% volume 10062 NUMERIC OPTIONAL volume of formamide to add to the dried pellet µl 2.5 2.5 SINGLE_STEP add H2O add 1 µl H2O volume 10063 NUMERIC OPTIONAL volume of H20 to add µl 1.0 1 SINGLE_STEP dissolve at 37°C dissolve at 37°C for 20 minutes and vortex in between. temperature 10064 NUMERIC OPTIONAL dissolving temperature °C 37.0 37 time 10065 NUMERIC OPTIONAL dissolving time minutes 20.0 20 SINGLE_STEP add 20xSSC add 20xSSC to a final concentration of 2xSSC volume 10066 NUMERIC OPTIONAL volume of 20xSSC to add µl 0.5 0.5 SINGLE_STEP add dextrane sulfate add 25% dextrane sulfate to a final concentration of 5% mix well before use volume 10067 NUMERIC OPTIONAL volume of 25% dextrane sulfate to add µl 1.0 1 SINGLE_STEP Denaturation of the DNA probe denaturate the hybridization mix (probe) - begin with denaturation while the cells are being treated with 2xSSC (see cell preparation) temperature 10068 NUMERIC OPTIONAL denaturation temperature °C 85.0 85 time 10069 NUMERIC OPTIONAL denaturation time minutes 10.0 10 SINGLE_STEP prehybridize the probe prehybridize the probe time 10070 NUMERIC OPTIONAL prehybridization time minutes 20.0 20 temperature 10071 NUMERIC OPTIONAL prehybridization temperature °C 37.0 37 STEP_GROUP Cell preparation cell are prepared for hybridization. - permeabilized - fixed - RNAse treated SINGLE_STEP cell permeabilization / extraction permeabilize / extract cells in CSK Buffer CSK BUffer: 100 mM NaCl 300 mM Sucrose 10 mM PIPES pH 6.8 3 mM MgCl2 0.5 % Triton-X 100 0.5 % DEPC (not really needed for DNA-FISH) 2 mM VRC (Vanadyl Ribonucleoside Complexes) (not really needed for DNA-FISH) temperature 10072 NUMERIC OPTIONAL extraction temperature °C 4.0 4 time 10073 NUMERIC OPTIONAL extraction time minutes 1.0 1 SINGLE_STEP cell fixation fix the cells after permeabilization / extraction in 3.5% PFA/PBS-- time 10074 NUMERIC OPTIONAL fixation time minutes 25.0 25 temperature 10075 NUMERIC OPTIONAL fixation temperature °C 25.0 25 SINGLE_STEP wash in PBS-- wash the fixed cells in PBS-- after fixation, to remove the remaining PFA time 10076 NUMERIC OPTIONAL washing time minutes 5.0 5 repeats 10077 NUMERIC OPTIONAL washing step repeats 2.0 2 temperature 10078 NUMERIC OPTIONAL washing temperature °C 25.0 25 SINGLE_STEP store cells in 70% EtOH cells are in 70% EtOH for future use in FISH-experiment. The cells should then be rehydrated for 10 min. in PBS--. temperature 10079 NUMERIC OPTIONAL storing temperature °C 4.0 4 time 10080 NUMERIC OPTIONAL time that the cells were stored in EtOH days 2.0 2 SINGLE_STEP rehydrate the stored cells rehydrate the EtOH stored cells in PBS--. time 10081 NUMERIC OPTIONAL rehydration time minutes 10.0 10 temperature 10082 NUMERIC OPTIONAL rehydration temperature °C 25.0 25 SINGLE_STEP cut the coverslip into 1/4s cut the coverslip into 1/4s in order to save some reagents. SINGLE_STEP RNAse treatment treat the fixed cells with RNAse-A (25 µg/ml in PBS--) in order to get rid of the RNA (we want to detect DNA only) concentration 10083 NUMERIC OPTIONAL RNAse-A concentration in PBS-- µg/ml 25.0 25 time 10084 NUMERIC OPTIONAL RNAse treatment time minutes 20.0 20 temperature 10085 NUMERIC OPTIONAL RNAse treatment temperature °C 25.0 25 SINGLE_STEP wash in PBS-- wash the RNAse treated cells in PBS-- in order to remove the excessive RNAse time 10086 NUMERIC OPTIONAL washing time minutes 10.0 5 temperature 10087 NUMERIC OPTIONAL washing temperature °C 25.0 25 repeats 10088 NUMERIC OPTIONAL washing step repeats 3.0 3 SINGLE_STEP 2xSSC treatment treat the cells with 2xSSC (start denaturating the probe while treating the cells with 2xSSC) time 10089 NUMERIC OPTIONAL 2xSSC treatment time minutes 3.0 10 SINGLE_STEP 2xSSC / 70% Formamide treatment treat the cells with 2xSSC / 70% Formamide. Use deionized formamide! time 10090 NUMERIC OPTIONAL 2xSSC / 70% Formamide treatment time minutes 3.0 3 SINGLE_STEP heat denaturate the chromatin heat denaturate the chromatin in order to make the DNA accessible for probe hybridization. This step is performed on a heating plate (Grant Boekel). Place a microscope slide onto the plate and apply a drop of 70% Formamide / 2xSSC (ca. 200µl) onto it. Let it preheat to the denaturation temperature and then put the coverslip (1/4) upside down (cells down) onto it. time 10091 NUMERIC OPTIONAL denaturation time minutes 4.5 4.5 temperature 10092 NUMERIC OPTIONAL denaturation temperature °C 85.0 85 STEP_GROUP dehydrate the cells dehydrate the cells in a EtOH series. SINGLE_STEP wash with 70% EtOH wash with 70% EtOH time 10093 NUMERIC OPTIONAL washing time minutes 5.0 5 temperature 10094 NUMERIC OPTIONAL washing temperature °C 4.0 4 SINGLE_STEP wash with 90% EtOH wash with 90% EtOH time 10095 NUMERIC OPTIONAL washing time minutes 5.0 5 temperature 10096 NUMERIC OPTIONAL washing temperature °C 4.0 4 SINGLE_STEP wash with 100% EtOH wash with 100% EtOH time 10097 NUMERIC OPTIONAL washing time minutes 5.0 5 temperature 10098 NUMERIC OPTIONAL washing temperature °C 4.0 4 SINGLE_STEP dry the cells at RT dry the cells at room temperature fo 5 min. time 10099 NUMERIC OPTIONAL drying time minutes 5.0 5 temperature 10100 NUMERIC OPTIONAL drying temperature °C 25.0 25 STEP_GROUP Hybridization Hybridization over night at 37°C in humidified chamber SINGLE_STEP apply the probe to the dehydrated cells apply the probe to the dehydrated cells, by pipetting the 5 µl onto a glass slide and putting the coverslip upside down onto the probe drop. Avoid bubbles! SINGLE_STEP seal with rubber cement seal with rubber cement SINGLE_STEP hybridize over 2 nights hybridize over 2 nights at 37° C in a humidified chamber. time 10101 NUMERIC OPTIONAL hybridization time hours 24.0 24 temperature 10102 NUMERIC OPTIONAL hybridization temperature °C 37.0 37 STEP_GROUP Washes after Hybridization Stringency washes after hybridization SINGLE_STEP 50% Formamide / 2xSSC washes 50% Formamide / 2xSSC washes preheat the 50% Formamide / 2xSSC solution to 45°C time 10103 NUMERIC OPTIONAL washing time minutes 15.0 15 temperature 10104 NUMERIC OPTIONAL washing temperature (preheat the solution to this temp.) °C 45.0 45 repeats 10105 NUMERIC OPTIONAL washing step repeat 2.0 2 SINGLE_STEP 0.1xSSC wash 0.1xSSC wash time 10106 NUMERIC OPTIONAL washing time minutes 10.0 10 temperature 10107 NUMERIC OPTIONAL washing temperature °C 25.0 25 SINGLE_STEP 2xSSC wash 2xSSC wash time 10108 NUMERIC OPTIONAL washing time minutes 10.0 10 temperature 10109 NUMERIC OPTIONAL washing temperature °C 25.0 25 SINGLE_STEP 4xSSC wash 4xSSC wash time 10110 NUMERIC OPTIONAL washing time minutes 5.0 5 temperature 10111 NUMERIC OPTIONAL washing temperature °C 25.0 25 STEP_GROUP Probe detection Detection of the hapten labelled probes using fluorescent antibodies. STEP_GROUP Biotin Probe detection detect hapten labeled probes by fluorescent antibodies SINGLE_STEP dilute streptavidin antibody dilute streptavidin antibody 1:200 in 4xSSC/0.1% BSA/0.01% Tween 20 dilution 10112 ENUMERATION OPTIONAL antibody dilution 1:200 1:200 1:200 antibody 10113 TEXT OPTIONAL type of antibody streptavidin, Alexa Fluor® 647 conjugate 2 mg/mL streptavidin, Alexa Fluor® 647 conjugate 2 mg/mL SINGLE_STEP apply the antibody solution to the cells apply the antibody solution to the cells by pipetting ~15 µl onto a glass slide and putting the coverslip upside down onto the antibody solution. Avoid air bubbles! SINGLE_STEP Incubate incubate for 1 hour at 37°C in a dark humidified chamber. time 10114 NUMERIC OPTIONAL incubation time hours 1.0 1 temperature 10115 NUMERIC OPTIONAL incubation temperature °C 37.0 37 STEP_GROUP Washes after Biotin-probe detection Washes after Biotin-probe detection SINGLE_STEP 4xSSC/0.1% TWEEN20 wash 4xSSC/0.1% TWEEN20 wash time 10116 NUMERIC OPTIONAL washing time minutes 10.0 10 temperature 10117 NUMERIC OPTIONAL washing temperature °C 25.0 25 SINGLE_STEP 4xSSC wash 4xSSC wash time 10118 NUMERIC OPTIONAL washing time minutes 10.0 10 temperature 10119 NUMERIC OPTIONAL washing temperature °C 25.0 25 repeats 10120 NUMERIC OPTIONAL washing step repeats 2.0 2 SINGLE_STEP 2xSSC wash 2xSSC wash time 10121 NUMERIC OPTIONAL washing time minutes 10.0 10 temperature 10122 NUMERIC OPTIONAL washing temperature °C 25.0 25 SINGLE_STEP PBS-- wash PBS-- wash time 10123 NUMERIC OPTIONAL washing time minutes 5.0 5 temperature 10124 NUMERIC OPTIONAL washing temperature °C 25.0 25 STEP_GROUP DNP/DIG Probe detection detect hapten labeled probes by fluorescent antibodies SINGLE_STEP dilute the DNP and DIG antibodies dilute DNP and DIG antibodies 1:200 in 4%BSA/0.1% Tween 20/PBS-- dilution 10125 ENUMERATION OPTIONAL antibody dilution 1:200 1:200 1:200 DNP antibody 10126 TEXT OPTIONAL type of antibody anti-dinitrophenyl-KLH, rabbit IgG fraction, Alexa Fluor® 488 conjugate 2 mg/mL anti-dinitrophenyl-KLH, rabbit IgG fraction, Alexa Fluor® 488 conjugate 2 mg/mL DIG antibody 10127 TEXT OPTIONAL DIG antibody Anti-Digoxigenin-Rhodamine, Fab fragments (ROCHE) Anti-Digoxigenin-Rhodamine, Fab fragments (ROCHE) SINGLE_STEP apply the antibody solution to the cells apply the antibody solution to the cells by pipetting ~15 µl onto a glass slide and putting the coverslip upside down onto the antibody solution. Avoid air bubbles! SINGLE_STEP Incubate incubate for 1 hour at room temperature in a dark humidified chamber. time 10128 NUMERIC OPTIONAL incubation time hours 1.0 1 temperature 10129 NUMERIC OPTIONAL incubation temperature °C 25.0 25 STEP_GROUP Washes after DNP/DIG-probe detection Washes after DNP/DIG-probe detection SINGLE_STEP PBS-- wash PBS-- wash time 10130 NUMERIC OPTIONAL washing time minutes 5.0 5 temperature 10131 NUMERIC OPTIONAL washing temperature °C 25.0 25 repeats 10132 NUMERIC OPTIONAL washing step repeats 3.0 3 SINGLE_STEP DAPI conterstain DAPI conterstain time 10133 NUMERIC OPTIONAL staining time minutes 5.0 5 temperature 10134 NUMERIC OPTIONAL staining temperaure °C 25.0 25 concentration 10135 NUMERIC OPTIONAL DAPI concentration in PBS-- µg/ml 0.05 0.05 SINGLE_STEP Mount and image Mount with an antifade mounting solution (Slow Fade Gold, Invitrogen) and seal with nail polish. Image on Epifluorescence microscope upload deadline upload deadline on May 1, 2009