Matt:LabNotes/2015-6-15: Difference between revisions

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==Motor Neurons RNA FISH Dye Coupling==
==Motor Neurons RNA FISH Dye Coupling==
*[[Matt:LabNotes/2015-6-8#DARTFISH]]
*[[Matt:LabNotes/2015-6-8#RNA_FISH | Previous RNA FISH attempt detected no signal]]
**Try again with higher expressed genes (CUX2 and KIT)
 
===Probe Resuspension===
**[[Media:GAO_022315.xlsx | Table of ordered plates]]
***KIT: Plate=KIT-SNAP25-20150128 Wells=A1-D12
***SNAP25: Plate=KIT-SNAP25-20150128 Wells=E1-H12
 
#Resuspend each oligo in 15 uL nuclase-free H2O (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 450 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
#*Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower...
#*Goal is to get 1ug/ul DNA concentration
#Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy<!--
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''measured (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''normalized (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Average'''
| align="center" style="background:#f0f0f0;"|'''Stdev'''
|-
| KIT 1:1||1228||1228||||
|-
| KIT 1:5||271.8||1359||||
|-
| KIT 1:10||135.4||1354||1313.7||74.2
|-
| SNAP25 1:1||1227.6||1227.6||||
|-
| SNAP25 1:5||258.5||1292.5||||
|-
| SNAP25 1:10||128.8||1288||1269.4||36.2
|-
|}
*For ADARB2 & CUX2 with approx 1300ng/ul
**Add 132ul H2O to 440ul
**1300ng/ul x 440ul = 1000ng/ul x 572ul -> 572-440=132ul H2O added
 
===Dye Coupling===
===Dye Coupling===
[[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br>
[[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br>

Revision as of 00:09, 16 June 2015

Motor Neurons RNA FISH Dye Coupling

Probe Resuspension

  1. Resuspend each oligo in 15 uL nuclase-free H2O (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 450 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
    • Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower...
    • Goal is to get 1ug/ul DNA concentration
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy