Daniel:Notebook/HiResChrPaint/2014-2-23: Difference between revisions

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'''Sample Matrix'''
'''Sample Matrix'''


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Sample
| width="91" | Sample A
| width="65" | uL sample A (pmol)
| width="65" | Sample B
| width="65" | uL sample B (pmol)


|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | V6S3 Only (A)
| [[Daniel:Notebook/HiResChrPaint/2014-2-21#Labeling Results|V6S3-546]]
| 5 (10)
| Unlabeled Cot1
| 1 (1 ug)
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | V6S3+Cot1 (B)
| [[Daniel:Notebook/HiResChrPaint/2014-2-21#Labeling Results|V6S3-546]]
| 5 (10)
| [[Daniel:Notebook/HiResChrPaint/2014-1-24#Labeling Results|Cot1-488]]
| 6 (10)
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | Multi-Color Cot1 (C)
| [[Daniel:Notebook/HiResChrPaint/2014-2-21#Labeling Results|Cot1-High-546]]
| 20 (4)
| [[Daniel:Notebook/HiResChrPaint/2014-2-21#Labeling Results|Cot1-Low-488]]
| 5 (5)
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="45" | BSPP Negative Control (D)
| [[Daniel:Notebook/HiResChrPaint/2014-2-21#Labeling Results|BSPP1]]
| 5 (10)
| Unlabeled Cot1
| 1 (1 ug)
|}


''Day 1''
''Day 1''


#Mix sample probes and vacuum centrifuge
#Allow stored samples to warm to room temperature
#Allow stored samples to warm to room temperature
#Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
#Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
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#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 overnight at 42C
 
[[Category:HRCP]] [[Category:20140208]] [[Category:Hybridization]]

Latest revision as of 18:26, 2 July 2014

Multi-Color FISH (Started 02/08/2014)[edit]

Back to Calendar

Hybridization[edit]

Sample Matrix

Sample Sample A uL sample A (pmol) Sample B uL sample B (pmol)
V6S3 Only (A) V6S3-546 5 (10) Unlabeled Cot1 1 (1 ug)
V6S3+Cot1 (B) V6S3-546 5 (10) Cot1-488 6 (10)
Multi-Color Cot1 (C) Cot1-High-546 20 (4) Cot1-Low-488 5 (5)
BSPP Negative Control (D) BSPP1 5 (10) Unlabeled Cot1 1 (1 ug)

Day 1

  1. Mix sample probes and vacuum centrifuge
  2. Allow stored samples to warm to room temperature
  3. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  4. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  5. Remove samples and allow to cool to room temperature
  6. Add 25 uL hybridization cocktail to center
  7. Add 22x22 #1.5 coverslip; seal with rubber cement
  8. Allow the rubber cement to air-dry for 5 minutes at room temperature
  9. Denature for 2.5 minutes at 92C; keep humid
  10. Transfer slides to a humidified chamber and hybridize overnight at 42C