Daniel:Notebook/HiResChrPaint/2014-7-15
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Strand Control (Started 07/08/2014)
Dye Labeling
I need to order more ULYSIS 488, so for now I'll just do 2 ug of V6S2 labeled with Alexa 546.
- Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
- For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
- VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
- Resuspend DNA in 20 uL labeling buffer (Component C)
- Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
- Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
- Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
- Purify using a column (Centri-Sep recommended)
Labeling Results
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye/probe | bp:dye ratio |
V6S2-546 | 60.2 | 3.5 | 2.3 | 1.6 | 52 |
V6S3-546 | 74.9 | 3.9 | 2.8 | 1.4 | 58 |
Some of my best results yet. Proceeding right to FISH.
Hybridization
Sample Matrix
Sample | Cot1-488 (ug) | V6S2-546 (pmol) | V6S3-546 (pmol) | Cot1 (ug) |
A | 0 | 8.7 (20) | 0 | 2.5 (2.5) |
B | 0 | 0 | 7.1 (20) | 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 37C or 42C