Daniel:Notebook/HiResChrPaint/2014-1-24
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Alu Control (Started 01/08/2014)[edit]
Cot1 Labeling[edit]
Labeling lots of Cot1 DNA for FISH experiments.
Samples:
- 5 ug Cot1 DNA
- The entire sample of V6S3-B
Protocol[edit]
1. 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! 2. Resuspend DNA in 20 uL labeling buffer (Component C) 3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly 4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL 5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath 5-1 Measure pre-column concentration using nanodrop 6. Purify using a Centri-Sep column
Labeling Results[edit]
Pre Column | Post Column | |||||||||
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | base:dye | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | base:dye | DNA retention (%) | Dye retention (%) |
Cot1 | 145.2 | 36.7 | 2.5 | 12 | 80.5 | 4.6 | 1.4 | 53 | 44.35 | 10.03 |
V6S3 | 76.4 | 24.6 | 2.9 | 9 | 57.7 | 0.9 | 2.2 | 194 | 60.42 | 2.93 |
These are great results, and I now have plenty of DNA for FISH.
FISH[edit]
Time to try FISH on the positive control.
Samples:
- 6 uL labeled Cot1 DNA (above)
- 10 uL V6S3 (above)
Original Protocol from Beliveau et al: FISH Protocol (Beliveau)
Day 1
0. Dry samples in vacuum centrifuge 1. Allow stored slides to warm to room temperature 2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media 3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes 4. Remove slides and allow to cool to room temperature 5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip 6. Invert slides on to cocktail-covered coverslips; seal with rubber cement 7. Allow the rubber cement to air-dry for 5 minutes at room temperature 8. Denature for 2.5 minutes at 92C; keep humid 9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C