Matt:LabNotes/2015-6-5: Difference between revisions

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>Mzcai
>Mzcai
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**SATB2
**SATB2
**KIT
**KIT
*Pick 2-3 genes and [[Daniel:Notebook/RNAFISH/2014-8-30#Oligo_Set_Preparations | resuspend]] if needed
*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]]


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#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 432 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
#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
  • Pick 2-3 genes and resuspend if needed
  1. Resuspend each oligo in 15 uL nuclase-free H2O (1 mM)
  2. Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
    1. Store the remaining oligos at -20C
  3. 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
  4. 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

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. 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!
  5. Add 8 uL sample to dye tube
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL nfH20 to sample
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  1. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  2. Remove supernatant and add 750 uL chilled 75% EtOH
  3. Centrifuge 5 minutes at 4 C
  4. Dry the pellet in the hood
  5. Resuspend pellet in 12 uL TE
  6. Check dye:probe ratio with Nanodrop