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==Motor Neurons RNA FISH Dye Coupling== | ==Motor Neurons RNA FISH Dye Coupling== | ||
*[[Matt:LabNotes/2015-6-8# | *[[Matt:LabNotes/2015-6-8#RNA_FISH | Previous RNA FISH attempt detected no signal]] | ||
**Try again with higher expressed genes (CUX2 and KIT) | |||
*[[Matt:LabNotes/2015-6-18 | RNA FISH continued here]] | |||
===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||1053.5||1053.5|||| | |||
|- | |||
| KIT 1:5||212.5||1062.5|||| | |||
|- | |||
| KIT 1:10||104.9||1049||1055||6.87 | |||
|- | |||
| SNAP25 1:1||1039.5||1039.5|||| | |||
|- | |||
| SNAP25 1:5||207.7||1038.5|||| | |||
|- | |||
| SNAP25 1:10||107.4||1074||1050.67||20.21 | |||
|- | |||
|} | |||
===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> | ||
Line 24: | Line 60: | ||
#*No vacufuge | #*No vacufuge | ||
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight | #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) | #Centrifuge at 4 C for 30 minutes (14,000 rpm) | ||
#Remove supernatant and add 750 uL chilled 75% EtOH | #Remove supernatant and add 750 uL chilled 75% EtOH | ||
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#*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) | #*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) | ||
#*Starting DNA mass = 5ul * 1ug/ul | #*Starting DNA mass = 5ul * 1ug/ul | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Probe''' | | align="center" style="background:#f0f0f0;"|'''Probe''' | ||
| align="center" style="background:#f0f0f0;"|'''ssDNA ng/ul''' | | align="center" style="background:#f0f0f0;"|'''ssDNA (ng/ul)''' | ||
| align="center" style="background:#f0f0f0;"|'''488 dye pmol/ul''' | | align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)''' | ||
| align="center" style="background:#f0f0f0;"|'''594 dye pmol/ul''' | | align="center" style="background:#f0f0f0;"|'''594 dye (pmol/ul)''' | ||
| align="center" style="background:#f0f0f0;"|''' | | 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;"|'''dye:probe ratio''' | ||
| align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | | align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | ||
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| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' | | 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 | |||
|} | |||
*[[Matt:LabNotes/2015-6-18 | RNA FISH continued here]] |
Latest revision as of 21:45, 18 June 2015
Motor Neurons RNA FISH Dye Coupling[edit]
- Previous RNA FISH attempt detected no signal
- Try again with higher expressed genes (CUX2 and KIT)
Probe Resuspension[edit]
- Table of ordered plates
- KIT: Plate=KIT-SNAP25-20150128 Wells=A1-D12
- SNAP25: Plate=KIT-SNAP25-20150128 Wells=E1-H12
- Table of ordered plates
- 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
' | measured (ng/ul) | normalized (ng/ul) | Average | Stdev |
KIT 1:1 | 1053.5 | 1053.5 | ||
KIT 1:5 | 212.5 | 1062.5 | ||
KIT 1:10 | 104.9 | 1049 | 1055 | 6.87 |
SNAP25 1:1 | 1039.5 | 1039.5 | ||
SNAP25 1:5 | 207.7 | 1038.5 | ||
SNAP25 1:10 | 107.4 | 1074 | 1050.67 | 20.21 |
Dye Coupling[edit]
Dan's best practice dye coupling protocol
Generic dye coupling protocol
- Sebastian said in email the two highest expressed genes from our 48 probe set of genes are CUX2 and KIT
- I am going to try both 488/594 and 546/647 pairs of fluorophores in case one set is not compatible with our Olympus filter cubes
CUX2-488 KIT-594 CUX2-546 KIT-647
- 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
- 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
Probe | ssDNA (ng/ul) | 488 dye (pmol/ul) | 594 dye (pmol/ul) | 546 dye (pmol/ul) | 647 dye (pmol/ul) | dye:probe ratio | ssDNA mass (ug) | ssDNA start mass (ug) | 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 |