Daniel:Notebook/RNAFISH/2014-8-30: Difference between revisions

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=Trial Run ([[Daniel:Notebokk/RNAFISH/2014-8-29|Started 08/29/2014]])=
=Trial Run ([[Daniel:Notebook/RNAFISH/2014-8-29|Started 08/29/2014]])=


[[Daniel:Notebook/RNAFISH|Back to Calendar]]
[[Daniel:Notebook/RNAFISH|Back to Calendar]]
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##20% for 1 h  
##20% for 1 h  
##30% at 4C overnight
##30% at 4C overnight
==Oligo Set Preparations==
These are the oligos that I ordered on  [[Daniel:Notebook/RNAFISH/2014-8-4|08/04/2014]].  Now that I am ready to do the experiment, I need to prep them.  First thing is to resuspend the oligos.  By order, each oligo has 15 nmol.
#Resuspend each oligo in 15 uL nfH2O (1 mM)
#Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
##Store the remaining oligos at -20C
#Add 432 uL nfH2O, diluting 10:1, for final concentration of 100 uM
#Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
===Nanodrop Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B7DEE8;font-size:12pt" align="center"
| width="65" height="33" | Sample
| width="65" | 1:1 Measured
| width="65" | 5:1 Measured
| width="65" | 5:1 Corrected
| width="65" | 10:1 Measured
| width="65" | 10:1 Corrected
|style="font-weight:bold" width="65" | Average
|style="font-weight:bold" width="65" | Standard Deviation
|- style="font-size:12pt"
| height="15"  valign="bottom" | GAD1
| align="center" align="center" | 558
| align="center" align="center" | 109.3
| align="center" align="center" | 546.5
| align="center" align="center" | 43.2
| align="center" align="center" | 432
|style="font-weight:bold" align="center" align="center" valign="bottom" | 512.2
| align="center" align="center" | 69.7
|- style="font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | FOXP2
|style="background-color:#D9D9D9" align="center" align="center" | 554.3
|style="background-color:#D9D9D9" align="center" align="center" | 114.9
|style="background-color:#D9D9D9" align="center" align="center" | 574.5
|style="background-color:#D9D9D9" align="center" align="center" | 54.5
|style="background-color:#D9D9D9" align="center" align="center" | 545
|style="background-color:#D9D9D9;font-weight:bold" align="center" align="center" valign="bottom" | 557.9
| align="center" align="center" | 15.1
|- style="font-size:12pt"
| height="15"  valign="bottom" | RBFOX3
| align="center" align="center" | 558.9
| align="center" align="center" | 115.2
| align="center" align="center" | 576
| align="center" align="center" | 56.8
| align="center" align="center" | 568
|style="font-weight:bold" align="center" align="center" valign="bottom" | 567.6
| align="center" align="center" | 8.6
|- style="font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | CUX2
|style="background-color:#D9D9D9" align="center" align="center" | 582.2
|style="background-color:#D9D9D9" align="center" align="center" | 124.9
|style="background-color:#D9D9D9" align="center" align="center" | 624.5
|style="background-color:#D9D9D9" align="center" align="center" | 60.6
|style="background-color:#D9D9D9" align="center" align="center" | 606
|style="background-color:#D9D9D9;font-weight:bold" align="center" align="center" valign="bottom" | 604.2
| align="center" align="center" | 21.2
|}
So each one has around 0.5 ug/uL.  Pretty good.
[[Category:RNAFISH]] [[Category:Hybridization]] [[Category:20140829]]

Latest revision as of 17:10, 24 September 2014

Trial Run (Started 08/29/2014)[edit]

Back to Calendar

Tissue Section Preparation (From 08/29/2014)[edit]

  1. Wash the tissue in PBS, 3 × 30 min
  2. Incubate the tissue in a sucrose series with increasing concentrations:
    1. 10% for 1 h
    2. 20% for 1 h
    3. 30% at 4C overnight

Oligo Set Preparations[edit]

These are the oligos that I ordered on 08/04/2014. Now that I am ready to do the experiment, I need to prep them. First thing is to resuspend the oligos. By order, each oligo has 15 nmol.

  1. Resuspend each oligo in 15 uL nfH2O (1 mM)
  2. Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
    1. Store the remaining oligos at -20C
  3. Add 432 uL nfH2O, diluting 10:1, for final concentration of 100 uM
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy

Nanodrop Results[edit]

Sample 1:1 Measured 5:1 Measured 5:1 Corrected 10:1 Measured 10:1 Corrected Average Standard Deviation
GAD1 558 109.3 546.5 43.2 432 512.2 69.7
FOXP2 554.3 114.9 574.5 54.5 545 557.9 15.1
RBFOX3 558.9 115.2 576 56.8 568 567.6 8.6
CUX2 582.2 124.9 624.5 60.6 606 604.2 21.2

So each one has around 0.5 ug/uL. Pretty good.