Daniel:Notebook/RNAFISH/2014-11-24

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Revision as of 19:06, 24 November 2014 by >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...")
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RNA FISH-Cell Lines

Back to Calendar

Hybridization

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)
  1. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  2. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  3. Drop temp to 65C
  4. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  5. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  6. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  7. 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