Daniel:Notebook/HiResChrPaint/2014-9-11

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Revision as of 21:52, 11 September 2014 by >Djacobse (→‎Hybridization)
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Padlock Probes II

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ARES Dye Labeling

I'm going to label a new dystrophin probe set for FISH. I'll use Alexa 488 for the probes.

Sample Matrix

Sample ng/uL uL added ug input
DMD-A 380 12.5 4.8
DMD-B 380 12.5 4.8
  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 2 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
  8. Recommend column purification after incubation

Labeling Results

Sample ng/uL dsDNA pmol/uL dye pmol/uL dsDNA pmol probe in 50 uL dye/probe bp:dye
DMD-A 55.9 9.3 8.5 423.5 1.1 18
DMD-B 54.2 8.9 8.2 410.6 1.1 18

ULYSIS Labeling

I'm going to label the padlock probes and some Cot1 with ULYSIS dye kits for FISH. I'll label the padlock probes with Alexa 488 and the Cot1 DNA with Alexa 546.

Sample Matrix

Sample ng/uL uL added ug input Dye uL dye
PPRL-A1 154.1 40 6.2 Alexa-488 5
PPRL-A2 131 40 5.2 Alexa-488 5
Cot1-A 1000 5 5 Alexa-546 5
Cot1-B 1000 5 5 Alexa-546 5
  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)

Labeling Results

Sample ng/uL dsDNA pmol/uL dye pmol/uL dsDNA pmol probe in 50 uL dye/probe bp:dye
PPRL-A1 92.6 2.7 1.4 70.2 1.9 52
PPRL-A2 93.8 2.8 1.4 71.1 2.0 51
Cot1-A 47.7 1.1 0.4 18.1 3.0 66
Cot1-B 50.7 1.1 0.4 19.2 2.9 70

Hybridization

Sample Matrix


  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 or 42C