Daniel:Notebook/HiResChrPaint/2013-9-27: Difference between revisions
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Using the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|Tuesday]], I'm going to do another FISH experiment. | Using the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|Tuesday]], I'm going to do another FISH experiment. | ||
'''Samples''': | |||
*10 pmol [[Daniel:Notebook/HiResChrPaint/2013-7-17#Labeling Results|V6S3]], 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-7-25#Labeling Results|V4S2]], 2.5 ug human Cot-1 DNA; | |||
*20 pmol [[Daniel:Notebook/HiResChrPaint/2013-9-5|BSPP-1-546]], 2.5 ug human Cot-1 DNA | |||
==FISH Protocol== | ==FISH Protocol== | ||
*Following [[Daniel:Protocols/FISH#Hybridization| | *Following modified [[Daniel:Protocols/FISH#Hybridization|FISH procedure]] | ||
''Day 1'' | ''Day 1'' | ||
1. Allow stored slides to warm to room temperature | 1. Allow stored slides to warm to room temperature | ||
2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C | 2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C; use pre-warmed solution | ||
3. Incubate in | 3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes | ||
4. Remove slides and allow to cool to room temperature | 4. Remove media from slides and allow to cool to room temperature | ||
5. Add 25 uL hybridization cocktail | 5. Add 25 uL hybridization cocktail to center of slide | ||
6 | 6. Denature for 2.5 minutes at 92C; keep humid; used rock bath | ||
7. Transfer slides to a humidified chamber and hybridize overnight at 42C | |||
Continued [[Daniel:Notebook/HiResChrPaint/2013-9-28|tomorrow]] | |||
Latest revision as of 16:56, 30 September 2013
FISH[edit]
Using the slides from Tuesday, I'm going to do another FISH experiment.
Samples:
- 10 pmol V6S3, 10 pmol V4S2, 2.5 ug human Cot-1 DNA;
- 20 pmol BSPP-1-546, 2.5 ug human Cot-1 DNA
FISH Protocol[edit]
- Following modified FISH procedure
Day 1
1. Allow stored slides to warm to room temperature 2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C; use pre-warmed solution 3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes 4. Remove media from slides and allow to cool to room temperature 5. Add 25 uL hybridization cocktail to center of slide 6. Denature for 2.5 minutes at 92C; keep humid; used rock bath 7. Transfer slides to a humidified chamber and hybridize overnight at 42C Continued tomorrow