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Daniel:Notebook/HiResChrPaint/2013-12-14
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=FISH Parameters= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] As discussed [[Daniel:Notebook/HiResChrPaint/2013-12-13|yesterday]], I'm going to explore several options in the FISH protocol for potential avenues. Dr. Zhang recommended yesterday that I use 96 well plates, like Hosuk, to explore these spaces. For now, since I've got the cells, I'm going to use the Mattek dishes, but especially with option 1 there is room to explore the space. Today I'm testing the protocol using low stringency conditions for #1, and if that yields noisy signal, as expected, then the best thing to do will be to explore the parameter space using various concentrations of formamide, for which I'll use a 96 well plate. '''Samples''' *Dish 3: 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-11-21#Labeling Results|V6S1/V6S2]]; 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-11-21#Labeling Results|V6S3/V6S4]]; 2.5 ug human Cot1 DNA *Dish 4: 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-11-21#Labeling Results|V6S1/V6S2]]; 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-11-21#Labeling Results|V6S3/V6S4]]; 2.5 ug human Cot1 DNA ==Hybridization== ''Original Protocol from Beliveau et al'': [[Media:Interphase_FISH_protocol.pdf|FISH Protocol (Beliveau)]] ''Day 1'' {| class="wikitable" |- |style="font-weight:bold" width="30" align="center"| '''Step''' |style="font-weight:bold" width="300" align="center"|'''Dish 3''' |style="font-weight:bold" width="300" align="center"|'''Dish 4''' |- |align="center"|1A. |align="center"|Allow stored slides to warm to room temperature |align="center"|Allow stored slides to warm to room temperature |- |align="center"|1A. |align="center"| |align="center"|Incubate 30 minutes in 70% Ethanol |- |align="center"|1B. |align="center"| |align="center"|Incubate 1 minutes in 0.1N HCl |- |align="center"|2. |align="center"|Incubate slides in 2X SSCT +25% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media |align="center"|Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media |- |align="center"|3. |align="center"|Incubate in 2X SSCT + 25% formamide at 60C for 20 minutes |align="center"|Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes |- |align="center"|4. |align="center"|Remove media and allow to cool to room temperature |align="center"|Remove media and allow to cool to room temperature |- |align="center"|5. |align="center"|Add 25 uL hybridization cocktail to center |align="center"|Add 25 uL hybridization cocktail to center |- |align="center"|6. |align="center"|Add a 22x22#1.5 coverslip to center of dish; seal with rubber cement |align="center"|Add a 22x22#1.5 coverslip to center of dish; seal with rubber cement |- |align="center"|7. |align="center"|Allow the rubber cement to air-dry for 5 minutes at room temperature |align="center"|Allow the rubber cement to air-dry for 5 minutes at room temperature |- |align="center"|8. |align="center"|Denature for 2.5 minutes at 92C; keep humid |align="center"|Denature for 2.5 minutes at 92C; keep humid |- |align="center"|9. |align="center"|Transfer slides to a humidified chamber and hybridize overnight at 37C |align="center"|Transfer slides to a humidified chamber and hybridize overnight at 37C |}
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