Daniel:Notebook/HiResChrPaint/2014-7-19: Difference between revisions

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>Djacobse
>Djacobse
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===Labeling Results===
===Labeling Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#BFBFBF;font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Sample
| width="65" | ng/uL ssDNA
| width="65" | pmol/uL dye
| width="71" | pmol/uL ssDNA
| width="65" | dye/probe
| width="71" | bp:dye ratio
|- style="font-size:12pt" align="center"
| height="30" | Dystrophin-488
| align="center" | 23.3
| align="center" | 3.9
| align="center" | 3.5
| align="center" | 1.1
| align="center" | 18
|- style="font-size:12pt" align="center"
| height="30" | Dystrophin-546
| align="center" | 25.2
| align="center" | 5.5
| align="center" | 3.8
| align="center" | 1.4
| align="center" | 14
|}
Good labeling results.  Proceeding to FISH.


==FISH Hybridization==
==FISH Hybridization==

Revision as of 22:07, 19 July 2014

Dystrophin Probes (Started 7/18/2014)

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Dye Labeling

  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 ssDNA pmol/uL dye pmol/uL ssDNA dye/probe bp:dye ratio
Dystrophin-488 23.3 3.9 3.5 1.1 18
Dystrophin-546 25.2 5.5 3.8 1.4 14

Good labeling results. Proceeding to FISH.

FISH Hybridization

  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 42C