Daniel:Notebook/HiResChrPaint/2014-7-1

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FISH w/ 70% Formamide (Started 06/30/2014)[edit]

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Dye Couple[edit]

  1. Dried samples in vacuum centrifuge
  2. 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!
  3. Resuspend DNA in 20 uL labeling buffer (Component C)
    1. Resuspended V6S2 in 80 uL labeling buffer, split into 4 tubes
  4. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
  5. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
    1. Added 1 uL 488 to each of Cot1 and V6S3
    2. Added 5 uL 546 to each of the 4 V6S2 samples
  6. Incubate at 80C for 25 minutes; stop the reaction by plunging the tube into an ice bath
  7. Purify using a column (Centri-Sep recommended)

Centri-Sep Column[edit]

  1. Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
  2. Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
  3. Drain the column into a wash tube (200-250 uL will drain)
  4. Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
  5. Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
  6. Place column into collection tube and centrifuge at 750xg for 2 minutes
  7. 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]

  1. Dried samples in vacuum centrifuge
    1. 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 1.5 uL 488 to each of Cot1 and V6S3; add 4 uL labeling buffer
  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)

Centri-Sep Column[edit]

  1. Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
  2. Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
  3. Drain the column into a wash tube (200-250 uL will drain)
  4. Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
  5. Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
  6. Place column into collection tube and centrifuge at 750xg for 2 minutes
  7. 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]

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)
  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 >24hrs at 42C