Daniel:Notebook/HiResChrPaint/2014-7-1
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FISH w/ 70% Formamide (Started 06/30/2014)[edit]
Dye Couple[edit]
- Cot1 (4ug), and V6S2/V6S3 from yesterday
- Dried samples in vacuum centrifuge
- 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)
- Resuspended V6S2 in 80 uL labeling buffer, split into 4 tubes
- 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
- Added 1 uL 488 to each of Cot1 and V6S3
- Added 5 uL 546 to each of the 4 V6S2 samples
- Incubate at 80C for 25 minutes; stop the reaction by plunging the tube into an ice bath
- Purify using a column (Centri-Sep recommended)
Centri-Sep Column[edit]
- Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
- Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
- Drain the column into a wash tube (200-250 uL will drain)
- Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
- Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
- Place column into collection tube and centrifuge at 750xg for 2 minutes
- Dry sample in vacuum centrifuge
Labeling Results[edit]
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye/probe | bp:dye ratio |
V6S2-546 | 54.4 | 2.2 | 1.6 | 1.4 | 75 |
V6S3-488 | 163.5 | 0.7 | 4.9 | 0.1 | 708 |
Cot1-488 | 50.3 | 1 | 0.5 | 2.0 | 152 |
Not great results, particularly for the 488 coupled reactions. I'm going to redo the labeling with the same sample, should enrich the 488 labeled DNA.
Dye Couple[edit]
- Dried samples in vacuum centrifuge
- 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 1.5 uL 488 to each of Cot1 and V6S3; add 4 uL labeling buffer
- Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
- Purify using a column (Centri-Sep recommended)
Centri-Sep Column[edit]
- Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
- Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
- Drain the column into a wash tube (200-250 uL will drain)
- Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
- Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
- Place column into collection tube and centrifuge at 750xg for 2 minutes
- Dry sample in vacuum centrifuge
Labeling Results[edit]
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye/probe | bp:dye ratio |
V6S3-488 | 209.8 | 3.9 | 6.2 | 0.6 | 163 |
Cot1-488 | 69.4 | 2.5 | 0.7 | 3.6 | 84 |
Better.
Hybridization[edit]
- Slides from Sunday 06/29/2014
Sample Matrix
Sample | Cot1-488 (ug) | V6S2-546 (pmol) | V6S3-488 (pmol) | BSPP1-546 (pmol) | Cot1 (ug) |
A | 0 | 9.4 (15) | 2.5 (15) | 0 | 2.5 (2.5) |
B | 0 | 9.4 (15) | 2.5 (15) | 0 | 2.5 (2.5) |
C | 25 (17.5) | 0 | 0 | 0 | 0 |
D | 0 | 0 | 0 | 5 (10) | 2.5 (2.5) |
- 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 >24hrs at 42C