Daniel:Notebook/HiResChrPaint/2014-10-2: Difference between revisions

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*Bienko Buffer
*Bienko Buffer
**5X Denhardt's solution
**5X Denhardt's solution
**1.7X SSC
**2X SSC
**50% Formamide
**50% Formamide
**10% dextran sulfate
**10% dextran sulfate
**50 mM Na2HPO4/NaH2PO4 buffer
*Raj Buffer
*Raj Buffer
**0.02% BSA
**0.02% BSA
**2X SSC
**2X SSC
**10% Formamide
**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==
==Hybridization==
'''Sample Matrix'''
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center"
| width="65" height="30" | Sample
| width="84" | Hyb. Buffer
| width="77" | uL DMD-B-488 (pmol)
| width="65" | uL Cot1-B-546 (ug)
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="30" | A
| Beliveau (Standard)
| 3.4 (30)
| 8 (0.4)
|- style="background-color:#D9D9D9;font-size:12pt" align="center"
|style="font-weight:bold" height="30" | B
| Beliveau (No Tween-20)
| 3.4 (30)
| 8 (0.4)
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="31" | C
| Bienko
| 3.4 (30)
| 8 (0.4)
|- style="background-color:#D9D9D9;font-size:12pt" align="center"
|style="font-weight:bold" height="29" | D
| Raj
| 3.4 (30)
| 8 (0.4)
|}

Revision as of 18:55, 2 October 2014

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)