Daniel:Notebook/HiResChrPaint/2014-7-15: Difference between revisions

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#Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
#Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
#Remove samples and allow to cool to room temperature
#Remove samples and allow to cool to room temperature
#Add 25 uL hybridization cocktail to center
#Add 25 uL hybridization cocktail to center + 1 uL RNase A
#Add 22x22 #1.5 coverslip; seal with rubber cement
#Add 22x22 #1.5 coverslip; seal with rubber cement
#Allow the rubber cement to air-dry for 5 minutes at room temperature
#Allow the rubber cement to air-dry for 5 minutes at room temperature
#Denature for 2.5 minutes at 92C; keep humid
#Denature for 2.5 minutes at 92C; keep humid
#Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C
#Transfer slides to a humidified chamber and hybridize >24 hrs at 42C
##Continued [[Daniel:Notebook/HiResChrPaint/2014-7-17|7/17/2014]]




[[Category:HRCP]] [[Category:20140708]] [[Category:ULYSIS Label]]
[[Category:HRCP]] [[Category:20140708]] [[Category:ULYSIS Label]] [[Category:Hybridization]]

Latest revision as of 17:24, 17 July 2014

Strand Control (Started 07/08/2014)[edit]

Back to Calendar

Dye Labeling[edit]

I need to order more ULYSIS 488, so for now I'll just do 2 ug of V6S2 labeled with Alexa 546.

  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[edit]

Sample ng/uL ssDNA pmol/uL dye pmol/uL ssDNA dye/probe bp:dye ratio
V6S2-546 60.2 3.5 2.3 1.6 52
V6S3-546 74.9 3.9 2.8 1.4 58

Some of my best results yet. Proceeding right to FISH.

Hybridization[edit]

Sample Matrix

Sample Cot1-488 (ug) V6S2-546 (pmol) V6S3-546 (pmol) Cot1 (ug)
A 0 8.7 (20) 0 2.5 (2.5)
B 0 0 7.1 (20) 2.5 (2.5)
C 5 (0.4) 0 0 0
  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 + 1 uL RNase A
  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 >24 hrs at 42C
    1. Continued 7/17/2014