Matt:LabNotes/2015-7-8: Difference between revisions
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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== | ===Combining CUX2_odd and CUX2_even=== | ||
#Oligos already resuspended in 15 uL nuclase-free H2O (1 mM) | #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) | #Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM) | ||
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|- | |- | ||
|} | |} | ||
*Added 55ul H2O to each (275ul) to make 330ul of 1000ng/ul | |||
==Dye Coupling== | |||
[[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> | |||
[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] | |||
CUX2-488 | |||
CUX2_odd-488 | |||
CUX2_even-594 | |||
SNAP25-594 | |||
#Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature | |||
#Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer | |||
#Denature samples for 5 minutes at 95C, then snap cool using ice box | |||
#Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing | |||
#*Dye cannot be saved for later use. Use immediately! | |||
#Add 8 uL sample to dye tube | |||
#Incubate in the dark for 1 hour | |||
#Add 10 uL 3M NaOAc and 80 uL 1xTE to sample | |||
#[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation | |||
#*Use 1X TE Buffer | |||
#*No vacufuge | |||
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight<!-- | |||
#Centrifuge at 4 C for 30 minutes (14,000 rpm) | |||
#Remove supernatant and add 750 uL chilled 75% EtOH | |||
#Centrifuge 5 minutes at 4 C | |||
#Dry the pellet in the hood | |||
#Resuspend pellet in 12 uL TE | |||
#Check dye:probe ratio with Nanodrop | |||
#*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) | |||
#*Starting DNA mass = 5ul * 1ug/ul | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''594 dye (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''546 dye (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''647 dye (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''dye:probe ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' | |||
|- | |||
| CUX2-488||227.7||39.1||0||N/A||N/A||1.11||2.7324||5||54.648 | |||
|- | |||
| KIT-594||251.2||-0.6||51.6||N/A||N/A||1.33||3.0144||5||60.288 | |||
|- | |||
| CUX2-546||246.9||N/A||N/A||43.7||0.2||1.15||2.9628||5||59.256 | |||
|- | |||
| KIT-647||268.6||N/A||N/A||6.8||45.6||1.10||3.2232||5||64.464 | |||
|}--> |
Revision as of 00:45, 9 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 | 1251.5 | 1251.5 | ||
CUX2_odd 1:5 | 247 | 1235 | ||
CUX2_odd 1:10 | 117.4 | 1174 | 1220 | 40.8 |
CUX2_even 1:1 | 1251.8 | 1251.8 | ||
CUX2_even 1:5 | 235.9 | 1179.5 | ||
CUX2_even 1:10 | 119.1 | 1191 | 1207 | 38.9 |
- Added 55ul H2O to each (275ul) to make 330ul of 1000ng/ul
Dye Coupling
Dan's best practice dye coupling protocol
Generic dye coupling protocol
CUX2-488 CUX2_odd-488 CUX2_even-594 SNAP25-594
- Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
- Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
- Dye cannot be saved for later use. Use immediately!
- Add 8 uL sample to dye tube
- Incubate in the dark for 1 hour
- Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
- Centri-Sep column purification after incubation
- Use 1X TE Buffer
- No vacufuge
- Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight