Daniel:Notebook/HiResChrPaint/2014-1-24

From ZhangLabWiki
Jump to navigation Jump to search

Alu Control (Started 01/08/2014)[edit]

Back to Calendar

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