Daniel:Notebook/RNAFISH/2014-10-11: Difference between revisions
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>Djacobse (Created page with "=ABP Trial Run (Started 10/10/2014)= Back to Calendar ==Dye Labeling-ARES== I need to make the appropriat...") |
>Djacobse |
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Dye | |style="font-weight:bold" height="15" valign="bottom" | Dye | ||
| align="center" valign="bottom" | Alexa-594 | | align="center" valign="bottom" | Alexa-594 | ||
| align="center" valign="bottom" | Alexa-488 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
Line 44: | Line 44: | ||
#Add 5 uL 3M NaOAc and 40 uL nfH20 to sample | #Add 5 uL 3M NaOAc and 40 uL nfH20 to sample | ||
#[[Daniel:Protocols/CentriSep|Centri Sep]] purification after incubation | #[[Daniel:Protocols/CentriSep|Centri Sep]] purification after incubation | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | |||
| width="83" height="30" | Sample | |||
| width="65" | ng/uL ssDNA | |||
| width="65" | pmol/uL dye | |||
| width="71" | pmol/uL ssDNA | |||
| width="71" | dye:probe | |||
| width="71" | bp:dye | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | FOXP2-594 | |||
| align="center" align="center" valign="bottom" | 61.3 | |||
| align="center" align="center" valign="bottom" | 11.9 | |||
| align="center" align="center" valign="bottom" | 9.3 | |||
| align="center" align="center" valign="bottom" | 1.3 | |||
| align="center" align="center" valign="bottom" | 16 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RBFOX3-488 | |||
| align="center" align="center" valign="bottom" | 54.9 | |||
| align="center" align="center" valign="bottom" | 9 | |||
| align="center" align="center" valign="bottom" | 8.3 | |||
| align="center" align="center" valign="bottom" | 1.1 | |||
| align="center" align="center" valign="bottom" | 18 | |||
|} | |||
As usual wit ARES, looks great. | |||
==Hybridization== | ==Hybridization== | ||
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##VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….) | ##VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….) | ||
##important to keep slides in a humid environment | ##important to keep slides in a humid environment | ||
===Buffers=== | |||
*Prehybridization solution: | |||
**50% deionized formamide | |||
**50% 2xSSC pH 7.0 | |||
**Add dextran sulfate to 1% | |||
*Hybridization solution A-Long Cai | |||
**2X SSC | |||
**20% Formamide | |||
**10% Dextran Sulfate | |||
*Hybridization solution B-Arjun Raj | |||
**2X SSC | |||
**10% Formamide | |||
**10% Dextran Sulfate | |||
**0.02% RNAse-free BSA | |||
**50 μg E.coli tRNA |
Latest revision as of 17:14, 14 October 2014
ABP Trial Run (Started 10/10/2014)[edit]
Dye Labeling-ARES[edit]
I need to make the appropriate probes, so I'll be labeling with the appropriate ARES dyes.
Sample | FOXP2 | RBFOX3 |
Dye | Alexa-594 | Alexa-488 |
ng/uL | 558 | 568 |
uL added | 10 | 10 |
ug added | 5.6 | 5.7 |
- Warm DMSO and bicarbonate labeling buffer and nuclease free water 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 solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 2 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Centri Sep purification after incubation
Labeling Results[edit]
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye:probe | bp:dye |
FOXP2-594 | 61.3 | 11.9 | 9.3 | 1.3 | 16 |
RBFOX3-488 | 54.9 | 9 | 8.3 | 1.1 | 18 |
As usual wit ARES, looks great.
Hybridization[edit]
- Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
- Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
- Drop temp to 65C
- Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
- Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
- Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
- Hybridize overnight appropriate temperature
- VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
- important to keep slides in a humid environment
Buffers[edit]
- Prehybridization solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- Hybridization solution A-Long Cai
- 2X SSC
- 20% Formamide
- 10% Dextran Sulfate
- Hybridization solution B-Arjun Raj
- 2X SSC
- 10% Formamide
- 10% Dextran Sulfate
- 0.02% RNAse-free BSA
- 50 μg E.coli tRNA