Daniel:Notebook/HiResChrPaint/2014-2-23: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 56: | Line 56: | ||
#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 |
Revision as of 19:54, 23 February 2014
Multi-Color FISH (Started 02/08/2014)
Hybridization
Sample Matrix
Sample | Sample A | uL sample A (pmol) | Sample B | uL sample B (pmol) |
V6S3 Only | V6S3-546 | 5 (10) | Unlabeled Cot1 | 1 (1 ug) |
V6S3+Cot1 | V6S3-546 | 5 (10) | Cot1-488 | 6 (10) |
Multi-Color Cot1 | Cot1-High-546 | 20 (4) | Cot1-Low-488 | 5 (5) |
BSPP Negative Control | 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