Daniel:Notebook/HiResChrPaint/2014-2-23: Difference between revisions
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>Djacobse (Created page with "=Multi-Color FISH (Started 02/08/2014)= Back to Calendar ==Hybridization== '''Sample Matrix''' ...") |
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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 | #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]
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
- Mix sample probes and vacuum centrifuge
- 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% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 42C