Daniel:Notebook/HiResChrPaint/2014-2-20
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Multi-Color FISH (Started 02/08/2014)[edit]
Time for a FISH run.
Samples
- V6S3-546 (+Cot1 unlabeled)
- V6S3-546 + Cot1-488
- Cot1 HighMW-546 + Cot1 LowMW-488
- BSPP-Either
These will be my samples. I also need to make slides and prep the samples, including dye coupling, so that will be today/tomorrow.
Sample Prep[edit]
Today, the main thing will be to pool my Cot1 High and Cot1 Low samples into two tubes and start to spin them down (they will need to be concentrated).
- Pool samples
- Measure in nanodrop
- Vacuum centrifuge for 1 hour
Dish Preparation[edit]
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
- Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI
- Allow cells to adhere for 6 hours at 37 C
- Rinse slides briefly in 1X PBS
- Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Rinse briefly in 1X PBS
- Incubate for 5 minutes in 1 mL of 2X SSCT
- Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
- Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
- Slides are typically best used within 1-2 weeks of creation