Daniel:Notebook/HiResChrPaint/2014-8-21: Difference between revisions

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>Djacobse
(Created page with "=Padlock Probes (Started 8/13/2014)= Back to Calendar ==Dye Labeling== Time for dye labeling ...")
 
>Djacobse
Line 5: Line 5:
==Dye Labeling==
==Dye Labeling==


Time for dye labeling of the padlock probes.
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.
 
#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'''!
#Resuspend DNA in 20 uL labeling buffer (Component C)
#Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
#Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
#Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
#Purify using a column (Centri-Sep recommended)
[[Image:ULYSIS_ReactionTable.png|500px]]

Revision as of 17:58, 21 August 2014

Padlock Probes (Started 8/13/2014)

Back to Calendar

Dye Labeling

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.

  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