Daniel:Notebook/HiResChrPaint/2014-7-1: Difference between revisions

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#Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
#Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
#Purify using a column (Centri-Sep recommended)
#Purify using a column (Centri-Sep recommended)
===Centri-Sep Column===
#Hydrate in 800 uL ddH20; Allow at least 30 minutes for hydration, be sure to hydrate all the gel
#Remove air bubbles by inverting and sharply tapping the column; stand up column and allow to settle
#Drain the column into a wash tube (200-250 uL will drain)
#Centrifuge at 750xg for 2 minutes (~3000 rpm; removes 300 uL fluid)
#Transfer 20 uL reaction mixture to the top of the gel; do not disturb gel surface
#Place column into collection tube and centrifuge at 750xg for 2 minutes
#Dry sample in vacuum centrifuge

Revision as of 18:36, 1 July 2014

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

Back to Calendar

Dye Couple

  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)

Centri-Sep Column

  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