Daniel:Notebook/HiResChrPaint/2014-2-20

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Revision as of 18:23, 2 July 2014 by >Djacobse
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Multi-Color FISH (Started 02/08/2014)[edit]

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Time for a FISH run.

Samples

  1. V6S3-546 (+Cot1 unlabeled)
  2. V6S3-546 + Cot1-488
  3. Cot1 HighMW-546 + Cot1 LowMW-488
  4. 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).

  1. Pool samples
  2. Measure in nanodrop
  3. Vacuum centrifuge for 1 hour

Dish Preparation[edit]

  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
  2. Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI
  3. Allow cells to adhere for 6 hours at 37 C
  4. Rinse slides briefly in 1X PBS
  5. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
  6. Rinse briefly in 1X PBS
  7. Incubate for 5 minutes in 1 mL of 2X SSCT
  8. Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
  9. Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
  10. Slides are typically best used within 1-2 weeks of creation