Daniel:Notebook/RNAFISH/2015-2-25: Difference between revisions
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>Djacobse (Created page with "=Linnarsson Protocol (Started 2/24/2015)= Back to Calendar ==Buffer Prep== *Hybridization Buffer **RNAse f...") |
>Djacobse |
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#Dry the pellet in the hood; protect from light | #Dry the pellet in the hood; protect from light | ||
#Resuspend pellet in 15 uL TE buffer | #Resuspend pellet in 15 uL TE buffer | ||
===Dye Labeling 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="45" valign="bottom" | | |||
|style="font-weight:bold" width="65" align="center" | Column ng/uL | |||
|style="font-weight:bold" width="65" align="center" | Column pmol/uL | |||
|style="font-weight:bold" width="65" align="center" | Total Column DNA (ug) | |||
|style="font-weight:bold" width="65" align="center" | Total Column dye (pmol) | |||
|style="font-weight:bold" width="65" align="center" | Column dye/probe | |||
|style="font-weight:bold" width="65" align="center" | Column Base:dye | |||
|style="font-weight:bold" width="65" align="center" | EtOH Precip ng/uL | |||
|style="font-weight:bold" width="65" align="center" | EtOH Precip pmol/uL | |||
|style="font-weight:bold" width="65" align="center" | EtOH total DNA (ug) | |||
|style="font-weight:bold" width="65" align="center" | EtOH total dye (pmol) | |||
|style="font-weight:bold" width="65" align="center" | EtOH dye/probe | |||
|style="font-weight:bold" width="65" align="center" | EtOH Base:dye | |||
|style="font-weight:bold" width="65" align="center" | EtOH DNA Yield (%) | |||
|style="font-weight:bold" width="65" align="center" | EtOH dye Yield (%) | |||
|style="font-weight:bold" width="78" align="center" | Stock Concentration (uM) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | FOXP2-488 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 6.8 | |||
| align="center" align="center" valign="bottom" | 2.4 | |||
| align="center" align="center" valign="bottom" | 680 | |||
| align="center" align="center" valign="bottom" | 1.9 | |||
| align="center" align="center" valign="bottom" | 11 | |||
| align="center" align="center" valign="bottom" | 90.6 | |||
| align="center" align="center" valign="bottom" | 11.4 | |||
| align="center" align="center" valign="bottom" | 1.4 | |||
| align="center" align="center" valign="bottom" | 171 | |||
| align="center" align="center" valign="bottom" | 0.8 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 56.63 | |||
| align="center" align="center" valign="bottom" | 25.15 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 11.4 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | GAD1-594 | |||
| align="center" align="center" valign="bottom" | 55.7 | |||
| align="center" align="center" valign="bottom" | 38.1 | |||
| align="center" align="center" valign="bottom" | 5.6 | |||
| align="center" align="center" valign="bottom" | 3810 | |||
| align="center" align="center" valign="bottom" | 4.5 | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 96.9 | |||
| align="center" align="center" valign="bottom" | 14.1 | |||
| align="center" align="center" valign="bottom" | 1.5 | |||
| align="center" align="center" valign="bottom" | 211.5 | |||
| align="center" align="center" valign="bottom" | 1.0 | |||
| align="center" align="center" valign="bottom" | 21 | |||
| align="center" align="center" valign="bottom" | 26.10 | |||
| align="center" align="center" valign="bottom" | 5.55 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 14.1 | |||
|} | |||
So this is a great result, and also shows that there may be a considerable amount of unlabeled dye left after the spin column. For purposes of purification and increasing concentration, I should probably always do an Ethanol precipitation. It will also probably be necessary in the future to concentrate the probes even more, that is dissolve each probe in 5-7 uL and use it all in a go. | |||
==Hybridization== | ==Hybridization== |
Latest revision as of 21:42, 25 February 2015
Linnarsson Protocol (Started 2/24/2015)[edit]
Buffer Prep[edit]
- Hybridization Buffer
- RNAse free water 5.3 mL
- SSC 20X 1 mL
- Dextran sulfate 2 mL
- Formamide 1 mL
- E coli tRNA 500 uL
- RVC 200 mM (warm to 37) 100 uL
- BSA 50 mg/mL 40 uL
Ethanol Precipitation[edit]
- Centrifuge at 4 C for 30 minutes
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge at minutes at 4 C for 10 minutes
- Dry the pellet in the hood; protect from light
- Resuspend pellet in 15 uL TE buffer
Dye Labeling Results[edit]
Column ng/uL | Column pmol/uL | Total Column DNA (ug) | Total Column dye (pmol) | Column dye/probe | Column Base:dye | EtOH Precip ng/uL | EtOH Precip pmol/uL | EtOH total DNA (ug) | EtOH total dye (pmol) | EtOH dye/probe | EtOH Base:dye | EtOH DNA Yield (%) | EtOH dye Yield (%) | Stock Concentration (uM) | |
FOXP2-488 | 24 | 6.8 | 2.4 | 680 | 1.9 | 11 | 90.6 | 11.4 | 1.4 | 171 | 0.8 | 24 | 56.63 | 25.15 | 11.4 |
GAD1-594 | 55.7 | 38.1 | 5.6 | 3810 | 4.5 | 4 | 96.9 | 14.1 | 1.5 | 211.5 | 1.0 | 21 | 26.10 | 5.55 | 14.1 |
So this is a great result, and also shows that there may be a considerable amount of unlabeled dye left after the spin column. For purposes of purification and increasing concentration, I should probably always do an Ethanol precipitation. It will also probably be necessary in the future to concentrate the probes even more, that is dissolve each probe in 5-7 uL and use it all in a go.
Hybridization[edit]
- Prepare fresh paraformaldehyde 4% and keep at RT
- Fix sections with 4% paraformaldehyde for 15 minutes at RT
- Rinse sections twice with RT PBS
- Permeabilize with 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.