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 m (→Dye Coupling) |
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**One sample with CUX2-Alexa488 and SNAP25-Alexa 594 | **One sample with CUX2-Alexa488 and SNAP25-Alexa 594 | ||
== | ==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
- First Try
- Second Try
- Probe dye coupling
- Third Try with cooled CCD camera
- Finally saw some signal using CUX2-Alexa488 48 probes with Sebastian's motor neurons
- Camera did not have DAPI filters so did not use DAPI
- Only used one probe set per sample
- Samples are iNGN Bipolar Neuron's from Alex at Harvard
- Fixed 6/30/2015 and stored in 70% EtOH at 4C for ~9days before RNA FISH
- RNA-Seq FPKM data
- RNA FISH 48 Probes available
- CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25
- ADARB2 not in DARTFISH CA12kNov2014_V4
- CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25
- 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
- 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
' | 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 |