Daniel:Notebook/HiResChrPaint/2014-8-21: Difference between revisions

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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)
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#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)

Back to Calendar

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
  1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C)
    1. For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C)
    2. VORTEX THESE SOLUTIONS VIGOROUSLY BEFORE USE EVERY TIME!
  2. Resuspend DNA in 20 uL labeling buffer (Component C)
  3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly
  4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL
  5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath
  6. 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