Daniel:Notebook/HiResChrPaint/2014-8-21

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Padlock Probes (Started 8/13/2014)[edit]

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Dye Labeling[edit]

Time for dye labeling of the padlock probes. I'm going to label the padlock probes with Alexa 488 and I'm going to make a Cot1-546 for controls.

Sample Matrix

Sample uL Sample ug Sample uL ULS 488 uL ULS 546
Cot1 2 2 0 5
PPRL 40 1.4 2 0
  1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
    1. For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
    2. 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
  6. Purify using a column (Centri-Sep recommended)
File:ULYSIS ReactionTable.png

Labeling Results[edit]

Sample ng/uL ssDNA pmol/uL dye pmol/uL DNA dye/probe bp:dye
PPRL-488 33.5 0.6 1.0 0.6 169
Cot1-546 22.9 0.8 0.5 1.7 87

The results for PPRL are not very good, but it'll have to do. I should probably add more sample during hybridization.

FISH[edit]

Sample Matrix

Sample uL PPRL-488 (pmol) uL Cot1-546 (pmol) uL DMD-546 (pmol) uL Cot1-488 (ug)
A 25 (25) 25 (0.6) 0 0
B 0 0 10.5 (40) 6 (0.3)
  1. Allow stored samples 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 samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 22x22 #1.5 coverslip; 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