Matt:LabNotes/2015-6-5: Difference between revisions
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>Mzcai m (→Dye Coupling) |
>Mzcai |
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Line 13: | Line 13: | ||
**SATB2 | **SATB2 | ||
**KIT | **KIT | ||
*Pick 2-3 genes and [[Daniel:Notebook/RNAFISH/ | *Pick 2-3 genes and [[Daniel:Notebook/RNAFISH/2015-1-29 | resuspend]] if needed | ||
*Dr. Zhang suggested SLC6A1 and SATB2 | *Dr. Zhang suggested SLC6A1 and SATB2 | ||
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***SLC6A1: Plate=CUX2-SLC6A1-20150128 Wells=E1-H12 | ***SLC6A1: Plate=CUX2-SLC6A1-20150128 Wells=E1-H12 | ||
***SATB2: Plate=ADARB2-SATB2-20150128 Wells=E1-H12 | ***SATB2: Plate=ADARB2-SATB2-20150128 Wells=E1-H12 | ||
***Also resuspend CUX2 and ADARB2 for future | |||
**[[Media:RNAFISH_Genes_Info.xlsx | Gene information with CA12kNov14_V4 efficiencies]] | **[[Media:RNAFISH_Genes_Info.xlsx | Gene information with CA12kNov14_V4 efficiencies]] | ||
Line 24: | Line 25: | ||
#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) | ||
##Store the remaining oligos at -20C | ##Store the remaining oligos at -20C | ||
#Add | #Add 350 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... | #*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 and this is what Dan has found to get closest to that | |||
#Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy | #Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy | ||
Revision as of 23:52, 5 June 2015
RNA FISH: Probe Resuspension and Dye Coupling
Probe Resuspension
- The 5' amino modified (separated by C6) 48-probe set ordered by Dan:
- GAD1
- Already resuspended
- FOXP2
- Already resuspended
- CUX2
- Already resuspended
- SLC6A1
- ADARB2
- SATB2
- KIT
- GAD1
- Pick 2-3 genes and resuspend if needed
- Dr. Zhang suggested SLC6A1 and SATB2
- Table of ordered plates
- SLC6A1: Plate=CUX2-SLC6A1-20150128 Wells=E1-H12
- SATB2: Plate=ADARB2-SATB2-20150128 Wells=E1-H12
- Also resuspend CUX2 and ADARB2 for future
- Gene information with CA12kNov14_V4 efficiencies
- 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 350 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 and this is what Dan has found to get closest to that
- Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
Dye Coupling
Dan's best practice dye coupling protocol
Generic dye coupling protocol
SLC6A1-488
SATB2-594
- Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
- Add 5 uL DNA 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 nfH20 to sample
- Centri-Sep column purification after incubation
- Use 1X TE Buffer
- 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