Daniel:Notebook/RNAFISH/2015-3-5: Difference between revisions
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>Djacobse |
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===Nanodrop Results=== | ===Nanodrop Results=== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#E6B8B7;font-size:12pt" | |||
| align="center" width="65" height="30" valign="bottom" | | |||
| width="65" align="center" | ng/uL ssDNA | |||
| width="65" align="center" | pmol/uL dye | |||
| width="65" align="center" | ng/uL ssDNA | |||
| width="65" align="center" | pmol/uL dye | |||
| width="65" align="center" | dye/probe | |||
| width="65" align="center" | bp:dye ratio | |||
| width="65" align="center" | EtOH Yield (%) DNA | |||
| width="65" align="center" | EtOH Yield Dye (%) | |||
| width="65" align="center" | Total Yield DNA (%) | |||
| width="65" align="center" | uM Labeled DNA | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | FOXP2-488 | |||
| align="center" align="center" valign="bottom" | 23.5 | |||
| align="center" align="center" valign="bottom" | 6.7 | |||
| align="center" align="center" valign="bottom" | 230.3 | |||
| align="center" align="center" valign="bottom" | 40.0 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 73.50 | |||
| align="center" align="center" valign="bottom" | 44.78 | |||
| align="center" align="center" valign="bottom" | 34.55 | |||
| align="center" align="center" valign="bottom" | 34.9 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | GAD1-546 | |||
| align="center" align="center" valign="bottom" | 30.8 | |||
| align="center" align="center" valign="bottom" | 14.7 | |||
| align="center" align="center" valign="bottom" | 292.1 | |||
| align="center" align="center" valign="bottom" | 53.3 | |||
| align="center" align="center" valign="bottom" | 1.2 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 71.13 | |||
| align="center" align="center" valign="bottom" | 27.19 | |||
| align="center" align="center" valign="bottom" | 43.82 | |||
| align="center" align="center" valign="bottom" | 44.3 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | GAD1-594 | |||
| align="center" align="center" valign="bottom" | 52.2 | |||
| align="center" align="center" valign="bottom" | 31.6 | |||
| align="center" align="center" valign="bottom" | 351.7 | |||
| align="center" align="center" valign="bottom" | 66.1 | |||
| align="center" align="center" valign="bottom" | 1.2 | |||
| align="center" align="center" valign="bottom" | 16 | |||
| align="center" align="center" valign="bottom" | 50.53 | |||
| align="center" align="center" valign="bottom" | 15.69 | |||
| align="center" align="center" valign="bottom" | 52.76 | |||
| align="center" align="center" valign="bottom" | 53.3 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | FOXP2-647 | |||
| align="center" align="center" valign="bottom" | 28.3 | |||
| align="center" align="center" valign="bottom" | 27.7 | |||
| align="center" align="center" valign="bottom" | 311.2 | |||
| align="center" align="center" valign="bottom" | 54.0 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
| align="center" align="center" valign="bottom" | 17 | |||
| align="center" align="center" valign="bottom" | 82.47 | |||
| align="center" align="center" valign="bottom" | 14.62 | |||
| align="center" align="center" valign="bottom" | 46.68 | |||
| align="center" align="center" valign="bottom" | 47.2 | |||
|} | |||
==Hybridization== | ==Hybridization== |
Revision as of 18:50, 5 March 2015
Linnarsson Lab Protocol 2 (Started 3/4/2015)
Dye Coupling (Continued; Ethanol Precipitation)
- Remove from -80C
- Centrifuge at 4 C at 12000 rpm for 30 minutes
- Remove supernatant and add 700 uL chilled 75% EtOH
- Centrifuge at minutes at 4 C for 10 minutes at 12000 rpm
- Remove supernatant and dry the pellet in the hood
- Resuspend pellet in 7.5 uL TE buffer
- Store DNA at 4C for immediate use
Nanodrop Results
ng/uL ssDNA | pmol/uL dye | ng/uL ssDNA | pmol/uL dye | dye/probe | bp:dye ratio | EtOH Yield (%) DNA | EtOH Yield Dye (%) | Total Yield DNA (%) | uM Labeled DNA | |
FOXP2-488 | 23.5 | 6.7 | 230.3 | 40.0 | 1.1 | 17 | 73.50 | 44.78 | 34.55 | 34.9 |
GAD1-546 | 30.8 | 14.7 | 292.1 | 53.3 | 1.2 | 17 | 71.13 | 27.19 | 43.82 | 44.3 |
GAD1-594 | 52.2 | 31.6 | 351.7 | 66.1 | 1.2 | 16 | 50.53 | 15.69 | 52.76 | 53.3 |
FOXP2-647 | 28.3 | 27.7 | 311.2 | 54.0 | 1.1 | 17 | 82.47 | 14.62 | 46.68 | 47.2 |
Hybridization
Sample Matrix
- Prepare fresh paraformaldehyde 4% and keep at RT
- Fix sections with 4% paraformaldehyde for 15 minutes at RT
- Rinse sections twice with RT PBS
- Permeabilization-may use:
- Ethanol (70% EtOH, >90 minute incuabation at 4C)
- Rinse sections twice with RT PBS
- Prepare the wash buffer and let it sit at RT
- Warm hybridization buffer and warm it up to 37C
- Prepare hybridization reaction:
- hybridization buffer-100 uL; probe 2-10 uL
- Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.