Daniel:Notebook/RNAFISH/2014-12-15: 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:#B8CCE4;font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Sample
| width="65" | Cell Line
| width="78" | uL [[Daniel:Notebook/RNAFISH/2014-12-9|GAD1-488]] (ng)
| width="75" | uL [[Daniel:Notebook/RNAFISH/2014-12-9|CUX2-594]] (ng)
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | A2
| align="center" valign="bottom" | U87-MG
| align="center" valign="bottom" | 3 (85)
| align="center" valign="bottom" | 4 (86)
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | B2
| align="center" valign="bottom" | BE2-C
| align="center" valign="bottom" | 3 (85)
| align="center" valign="bottom" | 4 (86)
|}


#Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
#Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide

Latest revision as of 23:12, 15 December 2014

Cell Line RNA FISH 2[edit]

Back to Calendar

Since I have two more samples and not enough time to really do much else, I'm going to retry the cell line FISH.

Hybridization[edit]

Sample Matrix

Sample Cell Line uL GAD1-488 (ng) uL CUX2-594 (ng)
A2 U87-MG 3 (85) 4 (86)
B2 BE2-C 3 (85) 4 (86)
  1. Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
  2. Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
    1. Make solutions from absolute ethanol and depc/H20*.
  3. Dry in oven at 50-65C.
  4. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  5. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  6. Drop temp to 65C
  7. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  8. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  9. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  10. Hybridize overnight appropriate temperature
    1. VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
    2. 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