Preparatin f Metaphase Chrmsmes fr FISH (Prtcl frm the department f Human Genetics) ES culture shuld be sub-cnfluent and in an expnentially grwing phase. Change medium 4 hurs prir t start f experiment. Incubate ES cells with 100l f Clcemid (Rche - 10295892001; txic-handle and dispse accrding t safety guidelines) in 5 ml f medium fr 3 hurs in rder t arrest cells in the metaphase stage. Wash cells nce with PBS and then trypsinise cells as usual, cllect in a 15 ml falcn tube and spin dwn fr 5 minutes at 800 rpm Remve supernatant carefully Snap the tube t lsen the pellet (make sure t cmpletely lsen the pellet). Add 1ml f prewarmed (37 C) 0.56% KCl drpwise. Snap the pellet gently t get a unifrm suspensin (n pellet shuld be visible. If this is the case, pipette gently t get a hmgenus suspensin). Add an additinal 4ml 0.56% pre-warmed KCl and incubate fr 10 minutes at 37 C Centrifuge the tube at 800 rpm fr 5 minutes and remve the supernatant carefully. Snap the tube t lsen the pellet. Add 1-1.5 ml f ice-cld fixative (MeOH : Acetic acid ::3:1 prepared fresh and ice cld) t the abve tube. Transfer the cntents f the tube t a 1.5ml eppendrf tube. Centrifuge at 6000 rpm fr 1 minute and carefully remve the supernatant. Repeat this step tw t three times. Resuspend in fixative (abut 1ml) and this is nw ready t be drpped n slides. This preparatin can be stred at 20 C fr many years in fixative. Dip the slides in ice cld 40% methanl and allw t drip n a filter paper. Make sure the methanl cats the entire slide prperly after dipping. If uneven film is frmed, discard the slide and use a fresh slide repeating the prcedure again. With a gilsn pipette, drp 25-35l f cell suspensin n the slide frm a height f abut 10cm. As the fixative gradually evaprates, the surface f the slide becmes grainy. At this mment, place the slide face dwn int the steam f ht water bath (75 C) fr 5-10 secnds and then dry quickly by placing the slide n a metal plate. Incubate at 65 C vernight fr aeging. The slides can be stred dipped in 100% EtOH at 20 C until use
Labeling f prbes by Nick Translatin Labeled prbes can be stred fr lng perids at 20 C withut affecting the prbe quality. Hence large prbe amunts can be labeled at ne time. Reagents: Bitin-16-2 dexy-uridine-5 -triphsphate Cncentratin : 1mM Stre at 20 C Digxigenin-11-2 dexy-uridine-5 -triphsphate Cncentratin : 1mM Stre at 20 C -Mercaptethanl (Stck slutin) Cncentratin : 14.44 M/l Stre at 4 C Wrking Slutins: DNAse Stck cncentratin : 1mg/ml 5mg DNAse 2.5mg 0.3M NaCl 2.5mg Glycerine Make up the vlume t 5ml with aqua bidest. Stre at 20 C Wrking dilutin: Dilute 1l f the stck slutin in 1ml f ice-cld water immediately befre use. The vlume f the wrking stck used in the nick translatin reactin must be tested fr each new bath f DNAse I stck slutin. Carry ut a series f digestins with 2g f prbe DNA, 10l f 10x buffer, 10l f b-mercaptethanl, and 1l, 2l, 5l and 10l f the DNAse I dilutin respectively in a 100l reactin vlume. Incubate fr 2 hurs at 15 C. Thereafter, takn an aliqut f 5-10l and test the size f the digested DNA n a 1% agarse gel using a 1Kb ladder. Chse the vlume f DNAse that results in prbe fragments f 100-500nt in length fr the nick translatin reactin. The DNAse stck shuld be diluted immediately befre applying t the nick translatin reactin. dntp s Wrking dilutin: Diltute datp, dctp, dgtp and dttp each t 2mM. Add 25l f datp, dctp and dgtp and 5l f dttp and make up the vlume t 100l with aq.bidest Stre at 20 C 10x Nick translatin (NT) buffer 0.5M Tris HCl, ph7.5 50mM Magnesium Chlride 0.5mg/ml BSA Stre at 20 C
0.1M -Mercaptethanl 69l f 14.4M -mercaptethanl and make up the vlume t 10ml with aqua. Bidest Stre at 4 C Miscellaneus: G50 spin clumn 0.5M EDTA 10% SDS Prcedure: Cmbine as fllws: Fr Bitin labeling: xl DNA cntaining 2g DNA l 10x NT buffer 10 l 0.1M -Mercaptethanl 10 l dntp s 2l Bitin dutp xl DNAse l DNA plymerase Make up the vlume t 100l with sigma water Fr Digxigenin labeling: xl DNA cntaining 2g DNA l 10x NT buffer 10 l 0.1M -Mercaptethanl 10 l dntp s l Digxigenin dutp xl DNAse (diluted 1:100 fresh) l DNA plymerase Make up the vlume t 100 l with sigma water Mix the cntents by vrtexing and briefly spin Incubate fr 2hurs at 15 C Place the reactin n ice and keep it n ice until the actual size f the prbe mlecules is determined Take an aliqut f 5-10l, add gel lading buffer and lad it n a standard 1% agarse gel alng with a suitable size marker. The prbe mlecules shuld be visible as a smear. This shuld cntain nly fragments smaller than 500nt.and larger than 100nt. If this is the case, prceed with inactivatin. If the prbe is larger, add mre DNAse t the reactin kept n ice and incubate further abut 15-30 minutes and repeat the previus step. If part f the DNA is smaller than 100nt, repeat using a higher dilutin f DNAse
The reactin is inactivated by adding 3l f 0.5M EDTA (15mM final cncentratin), 1l f 10% SDS (0.1% final cncentratin), and heating it fr 15mins at 68 C. The labeled prbe can be separated frm the unincrprated nucletides by gel filtratin using a Sephadex G-50 spin clumn frm Amersham. The flw thrugh cntains nw the labeled prbe at a cncentratin f 20ng/l. The labeled prbe can be stred at 20 C r prceed t the DNA precipitatin step DNA Precipitatin Reagents: Muse-Ct1 DNA Stre at 20 C. Once thawed, stre at 4 C 3M Sdium acetate 40.824 gm Sdium acetate in 100ml water Adjust ph t 5.2 with acetic acid Stre at rm temperature r 4 C Prcedure: After the deactivatin f the Nick translatin reactin, d as fllws I. Add tgether fr single cpy sequences a. 200 ng f bitin labeled DNA (`15l if yield is gd) b. 200 ng f digxigenin labeled DNA (`15l if yield is gd) c. 1l Salmn sperm DNA (10g/ml) d. 1/10 th vlume 3M Sdium acetate e. 2.5 vlumes f 100% chilled Ethanl (-20 C) II. Add tgether fr repetitive sequences 50-200ng labeled DNA (`15l if yield is gd) 10g muse CtI DNA 1/10 th vlume 3M Sdium acetate 2.5 vlumes f 100% chilled Ethanl (-20 C Mix well Incubate vernight at 20 C (r fr 30 minutes at 80 C) Centrifuge at 13,000 rpm fr 30 minutes at 4 C Discard supernatant Wash 3x with 70% Ethanl Allw t dry at 37 C Reagents: Pretreatment f metaphase chrmsmes fr FISH
Pepsin Stck slutin is 10% pepsin Stre at 20 C Pepsin wrking dilutin 700l f 1M HCl in 70ml f Aq.bidest water. Warm t 37 C and just befre the slides are t be dipped, add 50l f pepsin and mix well 10x PBS 82g NaCl 2g KCl 2g KHPO4 11.5g NaHPO4 x 2H2O Make up the vlume t 1L with water Stre at rm temperature Wrking dilutin : 1x PBS PBS-Magnesium Chlride 5ml 1M Magnesium chlride (101.65gm MgCl2 in 500ml water) + 95ml 1x PBS Stre at rm temperature 37% Frmaldehyde 70%, 90% and 100% Ethanl 20x SSC 175.3g NaCl 88.2g Sdium Citrate.2H2O Adjust ph t 7.0 Adjust vlume t 1L with water Wrking dilutin: 2x SSC Denaturatin Mix Freshly prepare 100l 20x SSC 200l Aq.bidest 7l 1M HCl 700l de-inised frmamide Prcedure: This prcedure can be carried ut simultaneusly with the precipitated DNA being centrifuged Equilibrate the slide by briefly dipping in 2x SSC Digest in pre-warmed pepsin slutin (remember t add pepsin (50ul) nly befre incubating the slide!) and incubate at 37 C fr 10 minutes in a shaking water bath Wash 2x5 mins in 1x PBS at rm temperature n a shaker Wash fr 3 minutes in 1x PBS-Magnesium Chlride at rm temperature n a shaker
Pre-fixing: Incubate the slide in 70ml PBS-Magnesium chlride cntaining 2ml 37% Frmaldehyde fr 5 minutes at rm temperature (n shaking) Wash 5 minutes in 1x PBS n a shaker Dehydrate the slide by dipping in Ethanl series (3 minutes each in 70%, 90% and 100% Ethanl) Allw the slide t air dry Place the Ethanl series in 20 C. Pre-warm the slides t 60 C Once the slide is dry, wrking quickly, add 100l f pre-warmed (75 C) denaturatin mix n the area f the slide marked with gd metaphase chrmsmes, place a 24 x 50 mm cverslip ver the slide and incubate it in a steel cntainer with lid fr 1 minute 45 secnds at 72 C-75 C r n a heated PCR blck (75 C) Remve the cver slip immediately and dip the slide briefly in ice-cld 2x SSC Dip the slides in ice cld Ethanl series (3 minutes each in ice-cld 70%, 90% and 100% Ethanl) Allw the slide t air dry Slides are ready fr hybridizatin Wrking slutins: FISH De-inized frmamide Stre at 4 C Hybridizatin buffer Prepare 20x SSC and 50% dextran sulphate slutins. Disslve dextran sulphate thrughly, then autclave it r filter it thrugh nitrcellulse filter. Cmbine 200l f 20x SSC and 400l 50% dextran sulphate with 400 l water. Stre at 4 C Prcedure: T air dried precipitated DNA prbe, add 6l de-inized frmamide and incubate it fr abut 1 hur at 37 C in a thermshaker (under shaking cnditins) Add 6lf hybridizatin buffer and mix thrughly and carefully Cntinue incubatin withut shaking fr an additinal 15 minutes Denature the DNA by incubating fr 5 minutes at 75 C Place immediately n ice fr abut 5 minutes. (If the prbe is meant fr repetitive sequences, preanneal the DNA by incubating the prbe at 37 C fr 40 minutes) Place the prbe carefully n the slide area marked with gd metaphase chrmsmes and place a 18 x 18mm cverslip ver the prbe, seal arund the cver slip with rubber cement and place in a humidified chamber. Hybridize vernight at 37 C in a humidified incubatr.
DETECTION Reagents: Antifade Mwil with DABCO Stre at 4 C Sensitive t light!! Avidin-FITC Flurescein Avidin DCS Cncentratin: 2mg/ml Stre at -20 C, nce thawed, stre at 4 C Sensitive t light Anti-Avidin Anti-DIG Bitinilated Anti-Avidin D Cncentratin: 0.5mg/ml Stre at -20 C, nce thawed, stre at 4 C Mnclnal Anti-Digxin (muse ascites fluid) Stre at -20 C, nce thawed, stre at 4 C Anti-Muse Cy3 Anti-Muse Cy3 Stre at -20 C, nce thawed, stre at 4 C Sensitive t light DAPI 4 6-Dianidin-2-phenylindl-2HCl Stck cncentratin: 0.2mg/ml Wrking dilutin: 10l Stck slutin in 100ml distilled water Stre in a Cplin jar wrapped in Alu fil at 4 C. Can be re-used many times Sensitive t light!!! Tween 20 Plyxyethylensrbitan Mnlaurate Stre at rm temperature Wrking dilutins: Antibdy I Prepare fresh Centrifuge antibdy fr 5 minutes at 14,000rpm at 4C Detectin slutin 1000 l 200l 500l
Avidin-FITC 10l 2l 5l Antibdy 2 Prepare fresh Centrifuge antibdy fr 5 minutes at 13,000rpm at 4C Detectin slutin 1000 l 500l 600l Anti-Avidin 10l 5l 6l Anti-Dig 4l 2l 2.4l Antibdy 3 Prepare fresh Centrifuge antibdy fr 5 minutes at 13,000rpm at 4C Detectin slutin 1000 l 500l 600l Avidin-FITC 10l 5l 6l Anti-Muse-TRITC 20l 10l 12l Blcking Slutin Prepare fresh 0.09g BSA in 3 ml 4x SSC/Tween 20 slutin Detectin Slutin Blcking Slutin : 4x SSC/Tween 20 slutin ::1:2 20x SSC 20 X SSC: siehe Maniatis der Labrprtkll 4x SSC / Tween 20 140 ml 20x SSC + 560ml Distilled water Place n a magnetic stirrer and add 1.4ml Tween 20 Warm t 45 C 0.2x SSC 1ml 20x SSC + 99ml Distilled water Warm t 53 C Warm this first befre yu start yur experiment!! Prcedure: Remve the rubber cement and cver slip carefully and discard Incubate slide fr 10mins in 2x SSC at 37 C In a shaking water bath Incubate slide 2 x 7 mins in 0.2 x SSC at 53 C in a shaking water bath Briefly equilibrate slides in 4x SSC / Tween 20 Add 150l f blcking slutin n the cverslip, place the slide carefully ver the cverslip. Place the slide and cverslip in a rack with the cverslip facing dwn, place the rack in a humidified metal cntainer and incubate at 30 C fr 30 minutes
Carefully remve and discard the cverslip. Wash briefly in 4x SSC / Tween 20 (45 C) Incubate with 150l f antibdy I as befre at 37C fr 30 minutes Carefully remve and discard the cverslip. Wash 3 x 5 mins in 4x SSC / Tween 20 (45 C) Incubate with 150l f antibdy II as befre at 37C fr 45 minutes Carefully remve and discard the cverslip. Wash 3 x 5 mins in 4x SSC / Tween 20 (45 C) Incubate with 150l f antibdy III as befre at 37C fr 30 minutes Carefully remve and discard the cverslip. Wash 3 x 5 mins in 4x SSC / Tween 20 (45 C) Incubate fr 2-10 minutes in DAPI slutin in the dark Wash 5x in distilled water. Air dry in dark Add tw drps f anti-fade n the slide and place a 24 x 50 mm cverslip ver it Stre in dark at 4 C