Daniel:Notebook/HiResChrPaint/2014-7-19
Jump to navigation
Jump to search
Dystrophin Probes (Started 7/18/2014)[edit]
Dye Labeling[edit]
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 2 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Recommend column purification after incubation
Labeling Results[edit]
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye/probe | bp:dye ratio |
Dystrophin-488 | 23.3 | 3.9 | 3.5 | 1.1 | 18 |
Dystrophin-546 | 25.2 | 5.5 | 3.8 | 1.4 | 14 |
Good labeling results. Proceeding to FISH.
FISH Hybridization[edit]
Sample Matrix
Sample | Cot1-488 (ug) | Dystrophin-488 (pmol) | Dystrophin-546 (pmol) | Cot1 (ug) |
A | 0 | 8.6 (30) | 0 | 2.5 (2.5) |
B | 0 | 0 | 7.9 (30) | 2.5 (2.5) |
C | 5 (0.4) | 0 | 0 | 0 |
- Allow stored samples to warm to room temperature
- Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
- Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 42C