Daniel:Notebook/RNAFACS/2015-4-11

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In Tube FISH Test (Started 4/10/2015)[edit]

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Dye Labeling[edit]

  1. Centrifuge at 4 C for 30 minutes (14000 rpm)
  2. Remove supernatant and add 750 uL chilled 75% EtOH
  3. Centrifuge at minutes at 4 C
  4. Dry the pellet in the hood
  5. Resuspend pellet in 12 uL TE

Labeling Results[edit]

Probe ng/uL ssDNA pmol/uL dye total DNA (ug) dye/probe bp:dye ratio DNA Yield (%)
GAD1-488 96.4 16.0 1.4 1.1 18 64.84
RBFOX3-546 70.0 10.5 1.1 1.0 20 50.00
FOXP2-594 30.3 5.5 1.1 1.2 17 21.87

Hybridization[edit]

  1. Pellet nuclei at 1000xg at 4C for 5 minutes; remove supernatant
  2. Resuspend nuclei in 150 uL RPHB
  3. Split nuclei into 3 tubes (50 uL each; ~150K nuclei/tube)
  4. Add 1 uL labeled probes to 0.5-1ng/uL
  5. Incubate overnight at 30C