Daniel:Notebook/HiResChrPaint/2013-8-10: Difference between revisions

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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" | &nbsp;
|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]

Back to Calendar

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
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