Daniel:Notebook/HiResChrPaint/2014-10-2: Difference between revisions
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>Djacobse (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) | |||
**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 | |||
** | **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== | ==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]
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) |
- 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