Daniel:Notebook/HiResChrPaint/2014-8-21: Difference between revisions
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>Djacobse |
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Time for dye labeling of the padlock probes. I'm going to label the padlock probes with Alexa 488 and I'm going to make a Cot1-546 for controls. | Time for dye labeling of the padlock probes. I'm going to label the padlock probes with Alexa 488 and I'm going to make a Cot1-546 for controls. | ||
'''Sample Matrix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#E6B8B7;font-size:12pt" | |||
| width="65" height="30" valign="bottom" | Sample | |||
| width="65" align="center" | uL Sample | |||
| width="65" align="center" | ug Sample | |||
| width="65" align="center" | uL ULS 488 | |||
| width="65" align="center" | uL ULS 546 | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | Cot1 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 5 | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL | |||
| align="center" align="center" valign="bottom" | 40 | |||
| align="center" align="center" valign="bottom" | 1.4 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|} | |||
#Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C) | #Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C) | ||
Line 16: | Line 42: | ||
#Purify using a column (Centri-Sep recommended) | #Purify using a column (Centri-Sep recommended) | ||
[[Image:ULYSIS_ReactionTable.png|500px]] | [[Image:ULYSIS_ReactionTable.png|500px]] | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#D8E4BC;font-size:12pt" | |||
| width="65" height="30" valign="bottom" | Sample | |||
| width="65" align="center" | ng/uL ssDNA | |||
| width="65" align="center" | pmol/uL dye | |||
| width="65" align="center" | pmol/uL DNA | |||
| width="65" align="center" | dye/probe | |||
| width="65" align="center" | bp:dye | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-488 | |||
| align="center" align="center" valign="bottom" | 33.5 | |||
| align="center" align="center" valign="bottom" | 0.6 | |||
| align="center" align="center" valign="bottom" | 1.0 | |||
| align="center" align="center" valign="bottom" | 0.6 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 169 | |||
|- style="font-size:12pt" | |||
|style="background-color:#D9D9D9" height="15" valign="bottom" | Cot1-546 | |||
| align="center" align="center" valign="bottom" | 22.9 | |||
| align="center" align="center" valign="bottom" | 0.8 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 1.7 | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 87 | |||
|} |
Revision as of 21:56, 21 August 2014
Padlock Probes (Started 8/13/2014)
Dye Labeling
Time for dye labeling of the padlock probes. I'm going to label the padlock probes with Alexa 488 and I'm going to make a Cot1-546 for controls.
Sample Matrix
Sample | uL Sample | ug Sample | uL ULS 488 | uL ULS 546 |
Cot1 | 2 | 2 | 0 | 5 |
PPRL | 40 | 1.4 | 2 | 0 |
- 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
Labeling Results
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL DNA | dye/probe | bp:dye |
PPRL-488 | 33.5 | 0.6 | 1.0 | 0.6 | 169 |
Cot1-546 | 22.9 | 0.8 | 0.5 | 1.7 | 87 |