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" | &nbsp;
|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]

Back to Calendar

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]

  1. Centrifuge at 4 C for 30 minutes
  2. Remove supernatant and add 750 uL chilled 75% EtOH
  3. Centrifuge at minutes at 4 C for 10 minutes
  4. Dry the pellet in the hood; protect from light
  5. 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]

  1. Prepare fresh paraformaldehyde 4% and keep at RT
  2. Fix sections with 4% paraformaldehyde for 15 minutes at RT
  3. Rinse sections twice with RT PBS
  4. Permeabilize with ethanol (70% EtOH, >90 minute incuabation at 4C)
  5. Rinse sections twice with RT PBS
  6. Prepare the wash buffer and let it sit at RT
  7. Warm hybridization buffer and warm it up to 37C
  8. Prepare hybridization reaction:
    1. hybridization buffer-100 uL; probe 2-10 uL
  9. Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.