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

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
Line 31: Line 31:
[[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


<!--
<!--

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
  • 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 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...
  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