Matt:LabNotes/2015-6-5: Difference between revisions
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[[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> | ||
[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] | [[Daniel:Protocols/AlexaDyeCouple#ARES | 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 | |||
#[[Daniel:Protocols/CentriSep|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 | |||
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Revision as of 22:46, 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
- 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 432 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...
- 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