Daniel:Notebook/RNAFISH/2014-11-24: Difference between revisions
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>Djacobse (Created page with "=RNA FISH-Cell Lines= Back to Calendar ==Hybridization== Today I'm going to perform hybridization on the cell lines I fixed [[Daniel:Notebook/RN...") |
>Djacobse |
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| width="65" height="49" | Sample | | width="65" height="49" | Sample | ||
| width="65" | Cell Line | | width="65" | Cell Line | ||
| width="81" | uL RBFOX3-488 (ng) | | width="81" | uL [[Daniel:Notebook/RNAFISH/2014-10-11|RBFOX3-488]] (ng) | ||
| width="79" | uL FOXP2-594 (ng) | | width="79" | uL [[Daniel:Notebook/RNAFISH/2014-10-11|FOXP2-594]] (ng) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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#Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide | |||
#Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea. | |||
##Make solutions from absolute ethanol and depc/H20*. | |||
#Dry in oven at 50-65C. | |||
#Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution | #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. | #Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes. | ||
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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 | ||
*Prehybridization solution: | |||
**50% deionized formamide | |||
**50% 2xSSC pH 7.0 | |||
**Add dextran sulfate to 1% | |||
*Hybridization solution (Arjun Raj) | |||
**2X SSC | |||
**10% Formamide | |||
**10% Dextran Sulfate | |||
**0.02% RNAse-free BSA | |||
**50 μg E.coli tRNA |
Latest revision as of 22:28, 24 November 2014
RNA FISH-Cell Lines[edit]
Hybridization[edit]
Today I'm going to perform hybridization on the cell lines I fixed Saturday. I'll be using RBFOX3 (NeuN) and FOXP2 as my genes of choice.
Sample Matrix
Sample | Cell Line | uL RBFOX3-488 (ng) | uL FOXP2-594 (ng) |
A1 | BE2-C | 2 (110) | 2 (120) |
B1 | T98-G | 2 (110) | 2 (120) |
C1 | U-87MG | 2 (110) | 2 (120) |
D1 | NCI h1975 | 2 (110) | 2 (120) |
- Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
- Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
- Make solutions from absolute ethanol and depc/H20*.
- Dry in oven at 50-65C.
- 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
- Prehybridization solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- Hybridization solution (Arjun Raj)
- 2X SSC
- 10% Formamide
- 10% Dextran Sulfate
- 0.02% RNAse-free BSA
- 50 μg E.coli tRNA