Matt:LabNotes/2015-7-8: Difference between revisions

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>Mzcai
(Created page with "=iNGN Bipolar Neurons RNA FISH= * First Try * Second Try * Probe dye coupling *[[Matt:La...")
 
>Mzcai
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**One sample with CUX2-Alexa488 and SNAP25-Alexa 594
**One sample with CUX2-Alexa488 and SNAP25-Alexa 594


==Dye Coupling==
==Combining CUX2_odd and CUX2_even==
#Oligos already resuspended 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 230 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'''
|-
| CUX2_odd 1:1||1053.5||1053.5||||
|-
| CUX2_odd 1:5||212.5||1062.5||||
|-
| CUX2_odd 1:10||104.9||1049||1055||6.87
|-
| CUX2_even 1:1||1039.5||1039.5||||
|-
| CUX2_even 1:5||207.7||1038.5||||
|-
| CUX2_even 1:10||107.4||1074||1050.67||20.21
|-
|}

Revision as of 22:55, 8 July 2015

iNGN Bipolar Neurons RNA FISH

  • RNA FISH 48 Probes available
    • CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25
      • ADARB2 not in DARTFISH CA12kNov2014_V4
  • SNAP25 has highest FPKM value
  • CUX2 is low-medium expression but use as positive control because it was the only one that worked previously
    • One sample exact same as last time CUX2-Alexa488 with no DAPI
    • One sample with odd CUX2-Alexa488 and even CUX2-Alexa594 to check specificity (should colocalize or else non-specific binding)
    • One sample with CUX2-Alexa488 and SNAP25-Alexa 594

Combining CUX2_odd and CUX2_even

  1. Oligos already resuspended 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 230 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
' measured (ng/ul) normalized (ng/ul) Average Stdev
CUX2_odd 1:1 1053.5 1053.5
CUX2_odd 1:5 212.5 1062.5
CUX2_odd 1:10 104.9 1049 1055 6.87
CUX2_even 1:1 1039.5 1039.5
CUX2_even 1:5 207.7 1038.5
CUX2_even 1:10 107.4 1074 1050.67 20.21