Matt:LabNotes/2015-7-8: Difference between revisions

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#*Use 1X TE Buffer
#*Use 1X TE Buffer
#*No vacufuge
#*No vacufuge
#*Disturbed gel bed in CUX2_odd-488
#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)

Revision as of 03:25, 9 July 2015

iNGN Bipolar Neurons RNA FISH

  • RNA FISH 48 Probes available
    • CUX2, SLC6A1, ADARB2, SATB2, KIT, SNAP25
      • ADARB2 not in DARTFISH CA12kNov2014_V4
  • SNAP25 has highest FPKM value
  • CUX2 is low-medium expression but use as positive control because it was the only one that worked previously
    • One sample exact same as last time CUX2-Alexa488 with no DAPI
    • One sample with odd CUX2-Alexa488 and even CUX2-Alexa594 to check specificity (should colocalize or else non-specific binding)
    • One sample with CUX2-Alexa488 and SNAP25-Alexa 594

Combining CUX2_odd and CUX2_even

  1. Oligos already resuspended 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 230 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
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
' measured (ng/ul) normalized (ng/ul) Average Stdev
CUX2_odd 1:1 1251.5 1251.5
CUX2_odd 1:5 247 1235
CUX2_odd 1:10 117.4 1174 1220 40.8
CUX2_even 1:1 1251.8 1251.8
CUX2_even 1:5 235.9 1179.5
CUX2_even 1:10 119.1 1191 1207 38.9
  • Added 55ul H2O to each (275ul) to make 330ul of 1000ng/ul

Dye Coupling

Dan's best practice dye coupling protocol
Generic dye coupling protocol

CUX2-488 CUX2_odd-488 CUX2_even-594 SNAP25-594

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA (~1ug/ul) 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 1xTE to sample
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
    • Disturbed gel bed in CUX2_odd-488
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight