Daniel:Notebook/HiResChrPaint/2014-1-24: Difference between revisions
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>Djacobse |
>Djacobse |
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5-1 Measure pre-column concentration using nanodrop | 5-1 Measure pre-column concentration using nanodrop | ||
6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]] | 6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]] | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| align="center" width="65" height="15" | | |||
|style="background-color:#8DB4E2;font-weight:bold" width="65" colspan="4" | Pre Column | |||
|style="background-color:#E6B8B7;font-weight:bold" width="65" colspan="4" | Post Column | |||
| align="center" width="71" | | |||
| align="center" width="65" | | |||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
|style="background-color:#D9D9D9" height="45" | Sample | |||
|style="background-color:#8DB4E2" | ng/uL ssDNA | |||
|style="background-color:#8DB4E2" | pmol/uL dye | |||
|style="background-color:#8DB4E2" | pmol/uL ss probe | |||
|style="background-color:#8DB4E2" | base:dye | |||
|style="background-color:#E6B8B7" | ng/uL ssDNA | |||
|style="background-color:#E6B8B7" | pmol/uL dye | |||
|style="background-color:#E6B8B7" | pmol/uL ss probe | |||
|style="background-color:#E6B8B7" | base:dye | |||
|style="background-color:#D8E4BC" | DNA retention (%) | |||
|style="background-color:#D8E4BC" | Dye retention (%) | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#D9D9D9;font-weight:bold" height="15" | Cot1 | |||
|style="background-color:#8DB4E2" align="center" | 145.2 | |||
|style="background-color:#8DB4E2" align="center" | 36.7 | |||
|style="background-color:#8DB4E2" align="center" | 2.5 | |||
|style="background-color:#8DB4E2" align="center" | 12 | |||
|style="background-color:#E6B8B7" align="center" | 80.5 | |||
|style="background-color:#E6B8B7" align="center" | 4.6 | |||
|style="background-color:#E6B8B7" align="center" | 1.4 | |||
|style="background-color:#E6B8B7" align="center" | 53 | |||
|style="background-color:#D8E4BC" align="center" | 44.35 | |||
|style="background-color:#D8E4BC" align="center" | 10.03 | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#D9D9D9;font-weight:bold" height="15" | V6S3 | |||
|style="background-color:#8DB4E2" align="center" | 76.4 | |||
|style="background-color:#8DB4E2" align="center" | 24.6 | |||
|style="background-color:#8DB4E2" align="center" | 2.9 | |||
|style="background-color:#8DB4E2" align="center" | 9 | |||
|style="background-color:#E6B8B7" align="center" | 57.7 | |||
|style="background-color:#E6B8B7" align="center" | 0.9 | |||
|style="background-color:#E6B8B7" align="center" | 2.2 | |||
|style="background-color:#E6B8B7" align="center" | 194 | |||
|style="background-color:#D8E4BC" align="center" | 60.42 | |||
|style="background-color:#D8E4BC" align="center" | 2.93 | |||
|} | |||
These are great results, and I now have plenty of DNA |
Revision as of 22:49, 24 January 2014
Alu Control (Started 01/08/2014)
Cot1 Labeling
Labeling lots of Cot1 DNA for FISH experiments.
Samples:
- 5 ug Cot1 DNA
- The entire sample of V6S3-B
Protocol
1. 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! 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 5-1 Measure pre-column concentration using nanodrop 6. Purify using a Centri-Sep column
Labeling Results
Pre Column | Post Column | |||||||||
Sample | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | base:dye | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | base:dye | DNA retention (%) | Dye retention (%) |
Cot1 | 145.2 | 36.7 | 2.5 | 12 | 80.5 | 4.6 | 1.4 | 53 | 44.35 | 10.03 |
V6S3 | 76.4 | 24.6 | 2.9 | 9 | 57.7 | 0.9 | 2.2 | 194 | 60.42 | 2.93 |
These are great results, and I now have plenty of DNA