Daniel:Notebook/HiResChrPaint/2014-10-2

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Revision as of 18:55, 2 October 2014 by >Djacobse (→‎Hybridization Buffer Test)
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Hybridization Buffer Test

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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
  • Raj Buffer
    • 0.02% BSA
    • 2X SSC
    • 10% Formamide

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

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)