Daniel:Notebook/HiResChrPaint/2014-7-19: Difference between revisions
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#Add 5 uL 3M NaOAc and 40 uL nfH20 to sample | #Add 5 uL 3M NaOAc and 40 uL nfH20 to sample | ||
#Recommend column purification after incubation | #Recommend column purification after incubation | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#BFBFBF;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="30" | Sample | |||
| width="65" | ng/uL ssDNA | |||
| width="65" | pmol/uL dye | |||
| width="71" | pmol/uL ssDNA | |||
| width="65" | dye/probe | |||
| width="71" | bp:dye ratio | |||
|- style="font-size:12pt" align="center" | |||
| height="30" | Dystrophin-488 | |||
| align="center" | 23.3 | |||
| align="center" | 3.9 | |||
| align="center" | 3.5 | |||
| align="center" | 1.1 | |||
| align="center" | 18 | |||
|- style="font-size:12pt" align="center" | |||
| height="30" | Dystrophin-546 | |||
| align="center" | 25.2 | |||
| align="center" | 5.5 | |||
| align="center" | 3.8 | |||
| align="center" | 1.4 | |||
| align="center" | 14 | |||
|} | |||
Good labeling results. Proceeding to FISH. | |||
==FISH Hybridization== | |||
'''Sample Matrix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |||
|style="font-weight:bold" width="65" height="30" | Sample | |||
|style="text-decoration:underline;color:#0000FF" width="65" | [[Daniel:Notebook/HiResChrPaint/2014-1-24|Cot1-488 (ug)]] | |||
|style="font-weight:bold;text-decoration:none" width="65" | Dystrophin-488 (pmol) | |||
|style="font-weight:bold" width="65" | Dystrophin-546 (pmol) | |||
|style="font-weight:bold" width="65" | Cot1 (ug) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | A | |||
| align="center" | 0 | |||
| 8.6 (30) | |||
| align="center" | 0 | |||
| 2.5 (2.5) | |||
|- style="background-color:#D9D9D9;font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | B | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| 7.9 (30) | |||
| 2.5 (2.5) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | C | |||
| 5 (0.4) | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
|} | |||
#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 42C | |||
[[Category:HRCP]] [[Category:20140718]] [[Category:ARES Label]] [[Category:Hybridization]] |
Latest revision as of 22:11, 19 July 2014
Dystrophin Probes (Started 7/18/2014)[edit]
Dye Labeling[edit]
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
- Denature samples for 5 minutes at 95C, then snap cool using ice box
- Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
- dye cannot be saved for later use. Use immediately!
- Add 2 uL dye to sample
- Incubate in the dark for 1 hour
- Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
- Recommend column purification after incubation
Labeling Results[edit]
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ssDNA | dye/probe | bp:dye ratio |
Dystrophin-488 | 23.3 | 3.9 | 3.5 | 1.1 | 18 |
Dystrophin-546 | 25.2 | 5.5 | 3.8 | 1.4 | 14 |
Good labeling results. Proceeding to FISH.
FISH Hybridization[edit]
Sample Matrix
Sample | Cot1-488 (ug) | Dystrophin-488 (pmol) | Dystrophin-546 (pmol) | Cot1 (ug) |
A | 0 | 8.6 (30) | 0 | 2.5 (2.5) |
B | 0 | 0 | 7.9 (30) | 2.5 (2.5) |
C | 5 (0.4) | 0 | 0 | 0 |
- 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 42C