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]
- 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