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

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#Resuspend each oligo in 15 uL nfH2O (1 mM)
#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)
#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
#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
#Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy

Revision as of 23:15, 30 August 2014

Trial Run (Started 08/29/2014)

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Tissue Section Preparation (From 08/29/2014)

  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

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

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.