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

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>Mzcai
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#Check dye:probe ratio with Nanodrop
#Check dye:probe ratio with Nanodrop
#*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
#*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
#*Starting DNA mass = 5ul * 1ug/ul<!--
#*Starting DNA mass = 5ul * 1ug/ul
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Probe'''
| align="center" style="background:#f0f0f0;"|'''Probe'''
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| align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)'''
| align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)'''
| align="center" style="background:#f0f0f0;"|'''594 dye (pmol/ul)'''
| align="center" style="background:#f0f0f0;"|'''594 dye (pmol/ul)'''
| align="center" style="background:#f0f0f0;"|'''546 dye (pmol/ul)'''
| align="center" style="background:#f0f0f0;"|'''647 dye (pmol/ul)'''
| align="center" style="background:#f0f0f0;"|'''dye:probe ratio'''
| align="center" style="background:#f0f0f0;"|'''dye:probe ratio'''
| align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)'''
| align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)'''
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| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)'''
| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)'''
|-
|-
| CUX2-488||227.7||39.1||0||N/A||N/A||1.11||2.7324||5||54.648
| CUX2-488||197.2||33.1||0.5||1.09||2.37||5||47.3
|-
|-
| KIT-594||251.2||-0.6||51.6||N/A||N/A||1.33||3.0144||5||60.288
| CUX2_odd-488||244||40.6||0.6||1.08||2.93||5||58.6
|-
|-
| CUX2-546||246.9||N/A||N/A||43.7||0.2||1.15||2.9628||5||59.256
| CUX2_even-594||295.9||-0.8||57.3||1.26||3.55||5||71
|-
|-
| KIT-647||268.6||N/A||N/A||6.8||45.6||1.10||3.2232||5||64.464
| SNAP25-594||227.4||-0.6||47.5||1.36||2.73||5||54.6
|}-->
|}
 
==RNA FISH Procedure==

Revision as of 21:58, 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
  10. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  11. Remove supernatant and add 750 uL chilled 80% EtOH
  12. Centrifuge 5 minutes at 4 C
  13. Dry the pellet in the hood
  14. Resuspend pellet in 12 uL TE
  15. Check dye:probe ratio with Nanodrop
    • Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
    • Starting DNA mass = 5ul * 1ug/ul
Probe ssDNA (ng/ul) 488 dye (pmol/ul) 594 dye (pmol/ul) dye:probe ratio ssDNA mass (ug) ssDNA start mass (ug) ssDNA Yield (%)
CUX2-488 197.2 33.1 0.5 1.09 2.37 5 47.3
CUX2_odd-488 244 40.6 0.6 1.08 2.93 5 58.6
CUX2_even-594 295.9 -0.8 57.3 1.26 3.55 5 71
SNAP25-594 227.4 -0.6 47.5 1.36 2.73 5 54.6

RNA FISH Procedure