Daniel:Notebook/HiResChrPaint/2014-10-2: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
(4 intermediate revisions by the same user not shown) | |||
Line 20: | Line 20: | ||
**50% Formamide | **50% Formamide | ||
**10% dextran sulfate | **10% dextran sulfate | ||
**10ug RNase A | |||
*Raj Buffer | *Raj Buffer | ||
**0.02% BSA | **0.02% BSA | ||
**2X SSC | **2X SSC | ||
**10% Formamide | **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. | 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. | ||
Line 35: | Line 37: | ||
| width="65" height="30" | Sample | | width="65" height="30" | Sample | ||
| width="84" | Hyb. Buffer | | width="84" | Hyb. Buffer | ||
| width="77" | uL DMD-B-488 (pmol) | | width="77" | uL [[Daniel:Notebook/HiResChrPaint/2014-9-11|DMD-B-488]] (pmol) | ||
| width="65" | uL Cot1-B-546 (ug) | | width="65" | uL [[Daniel:Notebook/HiResChrPaint/2014-9-11|Cot1-B-546]] (ug) | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
Line 63: | Line 65: | ||
|} | |} | ||
#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