Daniel:Notebook/HiResChrPaint/2014-9-11: Difference between revisions
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I'm going to label a new dystrophin probe set for FISH. I'll use Alexa 488 for the probes. | I'm going to label a new dystrophin probe set for FISH. I'll use Alexa 488 for the probes. | ||
'''Sample Matrix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt" align="center" | |||
| width="65" height="39" | Sample | |||
| width="65" | ng/uL | |||
| width="65" | uL added | |||
| width="65" | ug input | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | DMD-A | |||
| align="center" align="center" valign="bottom" | 380 | |||
| align="center" align="center" valign="bottom" | 12.5 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 4.8 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | DMD-B | |||
| align="center" align="center" valign="bottom" | 380 | |||
| align="center" align="center" valign="bottom" | 12.5 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 4.8 | |||
|} | |||
#Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature | #Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature |
Revision as of 18:05, 11 September 2014
Padlock Probes II
ARES Dye Labeling
I'm going to label a new dystrophin probe set for FISH. I'll use Alexa 488 for the probes.
Sample Matrix
Sample | ng/uL | uL added | ug input |
DMD-A | 380 | 12.5 | 4.8 |
DMD-B | 380 | 12.5 | 4.8 |
- Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
- Add 2.5 uL DNA and 1.5 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 1 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
ULYSIS Labeling
I'm going to label the padlock probes and some Cot1 with ULYSIS dye kits for FISH. I'll label the padlock probes with Alexa 488 and the Cot1 DNA with Alexa 546.
Sample Matrix
Sample | ng/uL | uL added | ug input | Dye | uL dye |
PPRL-A1 | 154.1 | 40 | 6.2 | Alexa-488 | 5 |
PPRL-A2 | 131 | 40 | 5.2 | Alexa-488 | 5 |
Cot1-A | 1000 | 5 | 5 | Alexa-546 | 5 |
Cot1-B | 1000 | 5 | 5 | Alexa-546 | 5 |
- Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
- For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
- VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
- Resuspend DNA in 20 uL labeling buffer (Component C)
- Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
- Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
- Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
- Purify using a column (Centri-Sep recommended)
File:ULYSIS ReactionTable.png