Daniel:Notebook/HiResChrPaint/2014-10-2

From ZhangLabWiki
Jump to navigation Jump to search

Hybridization Buffer Test[edit]

Back to Calendar

I'm going to run a FISH test on several different hybridization buffers, from several protocols.

  • Beliveau Buffer (Standard)
    • 2X SSCT (0.1% Tween 20)
    • 10% dextran sulfate
    • 10 ug RNAse A
    • 50% Formamide
  • Beliveau Buffer (No Tween)
    • 2X SSC
    • 10% dextran sulfate
    • 10 ug RNAse A
    • 50% Formamide
  • Bienko Buffer
    • 5X Denhardt's solution
    • 2X SSC
    • 50% Formamide
    • 10% dextran sulfate
    • 10ug RNase A
  • Raj Buffer
    • 0.02% BSA
    • 2X SSC
    • 10% Formamide
    • 10ug RNase A

The original Bienko buffer called for 5X Denhardt's solution, 1.7X SSC, 70% Formamide, 10% dextran sulfate, 50 mM Na2HPO4/NaH2PO4 buffer, but this is not possible to make by dilution (requires you to make SSC and phosphate buffers by scratch and suspend them in formamide/dextran. The modified buffer can be made from simple dilutions.

Hybridization[edit]

Sample Matrix

Sample Hyb. Buffer uL DMD-B-488 (pmol) uL Cot1-B-546 (ug)
A Beliveau (Standard) 3.4 (30) 8 (0.4)
B Beliveau (No Tween-20) 3.4 (30) 8 (0.4)
C Bienko 3.4 (30) 8 (0.4)
D Raj 3.4 (30) 8 (0.4)
  1. Allow stored samples to warm to room temperature
  2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 22x22 #1.5 coverslip; seal with rubber cement
  7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  8. Denature for 2.5 minutes at 92C; keep humid
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C