Matt:LabNotes/2015-6-5: Difference between revisions
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| SLC6A1 1:10||147.4||1474||1444.1||76.8 | | SLC6A1 1:10||147.4||1474||1444.1||76.8 | ||
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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 | |||
*For SATB2 & SLC6A1 with approx 1400ng/ul | |||
**Add 176ul H2O to 440ul | |||
**1400ng/ul x 440ul = 1000ng/ul x 616ul -> 616-440=176ul H2O added | |||
===Dye Coupling=== | ===Dye Coupling=== |
Revision as of 18:44, 6 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
- GAD1
- Pick 2-3 genes and resuspend if needed
- Dr. Zhang suggested SLC6A1 and SATB2
- Table of ordered plates
- SLC6A1: Plate=CUX2-SLC6A1-20150128 Wells=E1-H12
- SATB2: Plate=ADARB2-SATB2-20150128 Wells=E1-H12
- Also resuspend CUX2 and ADARB2 for future
- Gene information with CA12kNov14_V4 efficiencies
- Table of ordered plates
- Resuspend each oligo in 15 uL nuclase-free H2O (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
- 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
- Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
' | measured (ng/ul) | normalized (ng/ul) | Average | Stdev |
ADARB2 1:1 | 1228 | 1228 | ||
ADARB2 1:5 | 271.8 | 1359 | ||
ADARB2 1:10 | 135.4 | 1354 | 1313.7 | 74.2 |
CUX2 1:1 | 1227.6 | 1227.6 | ||
CUX2 1:5 | 258.5 | 1292.5 | ||
CUX2 1:10 | 128.8 | 1288 | 1269.4 | 36.2 |
SATB2 1:1 | 1328.8 | 1328.8 | ||
SATB2 1:5 | 290 | 1450 | ||
SATB2 1:10 | 138.2 | 1382 | 1386.9 | 60.8 |
SLC6A1 1:1 | 1356.8 | 1356.8 | ||
SLC6A1 1:5 | 300.3 | 1501.5 | ||
SLC6A1 1:10 | 147.4 | 1474 | 1444.1 | 76.8 |
- 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
- For SATB2 & SLC6A1 with approx 1400ng/ul
- Add 176ul H2O to 440ul
- 1400ng/ul x 440ul = 1000ng/ul x 616ul -> 616-440=176ul H2O added
Dye Coupling
Dan's best practice dye coupling protocol
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
- 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
- Centrifuge at 4 C for 30 minutes (14,000 rpm)
- Remove supernatant and add 750 uL chilled 75% EtOH
- Centrifuge 5 minutes at 4 C
- Dry the pellet in the hood
- Resuspend pellet in 12 uL TE
- Check dye:probe ratio with Nanodrop