Daniel:Notebook/HiResChrPaint/2013-8-10: Difference between revisions
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>Djacobse (Created page with " ==Dye Coupling== *Following ULYSIS Protocol 1. Resuspend DNA in 20 uL labeling buffer (Component C) 2. Denature DNA for 5 minu...") |
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=S2 Probe Prep (ULS; [[Daniel:Notebook/HiResChrPaint/2013-8-8|Started 8/8/2013]])= | |||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | |||
==Dye Coupling== | ==Dye Coupling== | ||
I have 2 uL left of the dye, so I can only couple V4S2, which has ~2 ug left (you need 1 uL dye/ug DNA). | |||
0. Dry sample in vaccuum centrifuge | |||
*Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS Protocol]] | *Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS Protocol]] | ||
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1. Resuspend DNA in 20 uL labeling buffer (Component C) | 1. Resuspend DNA in 20 uL labeling buffer (Component C) | ||
2. Denature DNA for 5 minutes at 95C; snap cool on ice | 2. Denature DNA for 5 minutes at 95C; snap cool on ice | ||
3. Add 1 uL dye to sample | 3. Add 1 uL dye to sample per ug | ||
4. Incubate for 20 minutes at 80C, snap cool on ice | 4. Incubate for 20 minutes at 80C, snap cool on ice | ||
5. Purify using [[Daniel:Protocols/CentriSep|Centri Sep]] column | 5. Purify using [[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" | |||
| align="center" width="65" height="30" | | |||
|style="font-weight:bold" width="65" | ng/uL DNA | |||
|style="font-weight:bold" width="65" | pmol/uL dye | |||
|style="font-weight:bold" width="65" | ug DNA in 25 uL | |||
|style="font-weight:bold" width="65" | pmol/uL DNA | |||
|style="font-weight:bold" width="71" | dye/probe | |||
|style="font-weight:bold" width="71" | dye/100 bp | |||
|style="font-weight:bold" width="71" | Base:Dye Ratio (:1) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | V4S2 | |||
| align="center" | 85.1 | |||
| align="center" | 2 | |||
| align="center" | 2.1 | |||
| align="center" | 3.2 | |||
| align="center" | 0.6 | |||
| align="center" | 0.8 | |||
| align="center" | 129 | |||
|} |
Latest revision as of 01:43, 11 August 2013
S2 Probe Prep (ULS; Started 8/8/2013)[edit]
Dye Coupling[edit]
I have 2 uL left of the dye, so I can only couple V4S2, which has ~2 ug left (you need 1 uL dye/ug DNA).
0. Dry sample in vaccuum centrifuge
- Following ULYSIS Protocol
1. Resuspend DNA in 20 uL labeling buffer (Component C) 2. Denature DNA for 5 minutes at 95C; snap cool on ice 3. Add 1 uL dye to sample per ug 4. Incubate for 20 minutes at 80C, snap cool on ice 5. Purify using Centri Sep column
Labeling Results[edit]
ng/uL DNA | pmol/uL dye | ug DNA in 25 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
V4S2 | 85.1 | 2 | 2.1 | 3.2 | 0.6 | 0.8 | 129 |