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

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(Created page with "=Hybridization Buffer Test= Back to Calendar I'm going to run a FISH test on several different hybridization buffers, from several protocol...")
 
>Djacobse
 
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I'm going to run a FISH test on several different hybridization buffers, from several protocols.
I'm going to run a FISH test on several different hybridization buffers, from several protocols.


#Beliveau Buffer (Standard)
*Beliveau Buffer (Standard)
**2X SSCT (0.1% Tween 20)
**10% dextran sulfate
**10 ug RNAse A
**50% Formamide
*Beliveau Buffer (No Tween)
**2X SSC
**2X SSC
**10% dextran sulfate
**10% dextran sulfate
**10 ug RNAse A
**10 ug RNAse A
**50% Formamide
**50% Formamide
#Bienko Buffer
*Bienko Buffer
**
**5X Denhardt's solution
#Raj Buffer
**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==
==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 [[Daniel:Notebook/HiResChrPaint/2014-9-11|DMD-B-488]] (pmol)
| width="65" | uL [[Daniel:Notebook/HiResChrPaint/2014-9-11|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)
|}
#Allow stored samples to warm to room temperature
#Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
#Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
#Remove samples and allow to cool to room temperature
#Add 25 uL hybridization cocktail to center
#Add 22x22 #1.5 coverslip; seal with rubber cement
#Allow the rubber cement to air-dry for 5 minutes at room temperature
#Denature for 2.5 minutes at 92C; keep humid
#Transfer slides to a humidified chamber and hybridize overnight at 37C
[[Category:HRCP]] [[Category:Hybridization]] [[Category:20141002]]

Latest revision as of 21:49, 4 November 2014

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