Matt:LabNotes/2015-6-15: Difference between revisions

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#*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
#*Goal is to get 1ug/ul DNA concentration
#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
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*For ADARB2 & CUX2 with approx 1300ng/ul
**Add 132ul H2O to 440ul
**1300ng/ul x 440ul = 1000ng/ul x 572ul -> 572-440=132ul H2O added


===Dye Coupling===
===Dye Coupling===

Revision as of 01:39, 16 June 2015

Motor Neurons RNA FISH Dye Coupling

Probe Resuspension

  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 450 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
KIT 1:1 1053.5 1053.5
KIT 1:5 212.5 1062.5
KIT 1:10 104.9 1049 1055 6.87
SNAP25 1:1 1039.5 1039.5
SNAP25 1:5 207.7 1038.5
SNAP25 1:10 107.4 1074 1050.67 20.21

Dye Coupling

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

  • Sebastian said in email the two highest expressed genes from our 48 probe set of genes are CUX2 and KIT
  • I am going to try both 488/594 and 546/647 pairs of fluorophores in case one set is not compatible with our Olympus filter cubes

CUX2-488 KIT-594 CUX2-546 KIT-647

  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
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight