Matt:LabNotes/2015-6-5: Difference between revisions
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>Mzcai |
>Mzcai m (→Dye Coupling) |
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| SLC6A1 1:10||147.4||1474||1444.1||76.8 | | 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=== | ===Dye Coupling=== | ||
Line 74: | Line 80: | ||
#Add 8 uL sample to dye tube | #Add 8 uL sample to dye tube | ||
#Incubate in the dark for 1 hour | #Incubate in the dark for 1 hour | ||
#Add 10 uL 3M NaOAc and 80 uL | #Add 10 uL 3M NaOAc and 80 uL 1xTE to sample | ||
#[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation | #[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation | ||
#*Use 1X TE Buffer | #*Use 1X TE Buffer | ||
#*No vacufuge | |||
#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 | ||
Line 85: | Line 92: | ||
#Resuspend pellet in 12 uL TE | #Resuspend pellet in 12 uL TE | ||
#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) | |||
#*Starting DNA mass = 5ul * 1ug/ul | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA ng/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''488 dye pmol/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''594 dye pmol/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA pmol/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''dye:probe ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' | |||
|- | |||
| SLC6A1-488||247.8||43.3||0.6||38.18||'''1.13'''||2.97||5||'''59.4''' | |||
|- | |||
| SATB2-594||260.4||-0.6||52.6||40.12||'''1.31'''||3.12||5||'''62.4''' | |||
|} | |||
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Latest revision as of 15:31, 8 June 2015
RNA FISH: Probe Resuspension and Dye Coupling[edit]
Probe Resuspension[edit]
- 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[edit]
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 1xTE to sample
- Centri-Sep column purification after incubation
- Use 1X TE Buffer
- No vacufuge
- 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
- 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 | ssDNA pmol/ul | dye:probe ratio | ssDNA mass (ug) | ssDNA start mass (ug) | ssDNA Yield (%) |
SLC6A1-488 | 247.8 | 43.3 | 0.6 | 38.18 | 1.13 | 2.97 | 5 | 59.4 |
SATB2-594 | 260.4 | -0.6 | 52.6 | 40.12 | 1.31 | 3.12 | 5 | 62.4 |