Daniel:Notebook/HiResChrPaint/2014-10-2
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Hybridization Buffer Test
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) |