Daniel:Notebook/RNAFISH/2015-2-5: Difference between revisions
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==Hybridization== | ==Hybridization== | ||
'''Sample Matrix''' | |||
{| 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" | |||
| width="65" height="45" valign="bottom" | Sample | |||
| width="65" align="center" | uL FOXP2-488 | |||
| width="65" align="center" | uL GAD1-594 | |||
| width="87" align="center" | uL hybridization buffer | |||
| width="65" align="center" | uL formamide | |||
| width="65" align="center" | uL Total | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | A | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 20.5 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" | 25 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | B | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 18.5 | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" | 25 | |||
|} | |||
#Permeabilize by leaving at room temperature for 30 minutes in 0.6% Triton X-100/2xSSPEdepc | #Permeabilize by leaving at room temperature for 30 minutes in 0.6% Triton X-100/2xSSPEdepc |
Latest revision as of 23:45, 5 February 2015
Cell Line RNA FISH[edit]
Slide Preparation[edit]
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in culture media
- Add 20 uL of cell suspension per dish to the cener; add 80 uL culture media
- Allow cells to adhere for 6 hours at 37 C
- Rinse slides briefly in 1X PBS
- Fix slides for 5 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Wash 2x in 2xSSPE for 5 minutes each
Hybridization[edit]
Sample Matrix
Sample | uL FOXP2-488 | uL GAD1-594 | uL hybridization buffer | uL formamide | uL Total |
A | 1 | 1 | 20.5 | 2.5 | 25 |
B | 2 | 2 | 18.5 | 2.5 | 25 |
- Permeabilize by leaving at room temperature for 30 minutes in 0.6% Triton X-100/2xSSPEdepc
- Wash 2x for 5 minutes in 2X SSPE
- 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 37C
- 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