Matt:LabNotes/2015-6-15: Difference between revisions
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>Mzcai m (→Dye Coupling) |
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#*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 | ||
Line 68: | Line 67: | ||
#*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 | ||
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{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Probe''' | | align="center" style="background:#f0f0f0;"|'''Probe''' |
Revision as of 21:34, 16 June 2015
Motor Neurons RNA FISH Dye Coupling
- Previous RNA FISH attempt detected no signal
- Try again with higher expressed genes (CUX2 and KIT)
Probe Resuspension
- 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
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