Daniel:Notebook/RNAFACS/2015-4-11
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In Tube FISH Test (Started 4/10/2015)[edit]
Dye Labeling[edit]
- Centrifuge at 4 C for 30 minutes (14000 rpm)
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge at minutes at 4 C
- Dry the pellet in the hood
- 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]
- Pellet nuclei at 1000xg at 4C for 5 minutes; remove supernatant
- Resuspend nuclei in 150 uL RPHB
- Split nuclei into 3 tubes (50 uL each; ~150K nuclei/tube)
- Add 1 uL labeled probes to 0.5-1ng/uL
- Incubate overnight at 30C