Daniel:Notebook/HiResChrPaint/2014-1-24: Difference between revisions

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>Djacobse
(Created page with "=Alu Control (Started 01/08/2014)= Back to Calendar ==Cot1 Labeling== Labeling lots of Cot1 DNA...")
 
>Djacobse
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Labeling lots of Cot1 DNA for FISH experiments.
Labeling lots of Cot1 DNA for FISH experiments.


[[Daniel:Protocols/AlexaDyeCouple#ULYSIS|Following ULYSIS protocol]]
'''Samples''':
*5 ug Cot1 DNA
*The entire sample of [[Daniel:Notebook/HiResChrPaint/2014-1-13#Nanodrop Results]]
 
*[[Daniel:Protocols/AlexaDyeCouple#ULYSIS|Following ULYSIS protocol]]


  1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
  1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
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  4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
  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
  5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
  5-1 Add 5 uL nfH2O, mix thoroughly, then extract 5 uL and put into 45 uL nfH2O (pre-column sample)
  5-1 Measure pre-column concentration using nanodrop
  6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]]
  6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]]

Revision as of 22:37, 24 January 2014

Alu Control (Started 01/08/2014)

Back to Calendar

Cot1 Labeling

Labeling lots of Cot1 DNA for FISH experiments.

Samples:

1. 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!
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
5-1 Measure pre-column concentration using nanodrop
6. Purify using a Centri-Sep column