Daniel:Notebook/RNAFACS/2015-4-11: Difference between revisions
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>Djacobse |
>Djacobse |
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(3 intermediate revisions by the same user not shown) | |||
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#Dry the pellet in the hood | #Dry the pellet in the hood | ||
#Resuspend pellet in 12 uL TE | #Resuspend pellet in 12 uL TE | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" | |||
| width="96" height="30" valign="bottom" | Probe | |||
| width="65" align="center" | ng/uL ssDNA | |||
| width="65" align="center" | pmol/uL dye | |||
| width="85" align="center" | total DNA (ug) | |||
| width="65" align="center" | dye/probe | |||
| width="65" align="center" | bp:dye ratio | |||
| width="85" align="center" | DNA Yield (%) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | GAD1-488 | |||
| align="center" align="center" valign="bottom" | 96.4 | |||
| align="center" align="center" valign="bottom" | 16.0 | |||
| align="center" align="center" valign="bottom" | 1.4 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
| align="center" align="center" valign="bottom" | 18 | |||
| align="center" align="center" valign="bottom" | 64.84 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | RBFOX3-546 | |||
| align="center" align="center" valign="bottom" | 70.0 | |||
| align="center" align="center" valign="bottom" | 10.5 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
| align="center" align="center" valign="bottom" | 1.0 | |||
| align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 50.00 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | FOXP2-594 | |||
| align="center" align="center" valign="bottom" | 30.3 | |||
| align="center" align="center" valign="bottom" | 5.5 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
| align="center" align="center" valign="bottom" | 1.2 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 21.87 | |||
|} | |||
==Hybridization== | ==Hybridization== | ||
#Resuspend | #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 | #Incubate overnight at 30C |
Latest revision as of 23:00, 11 April 2015
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