Daniel:Notebook/HiResChrPaint/2013-9-5: Difference between revisions

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>Djacobse
(Created page with "=BSPP Control (Started 09/03/2013)= Back to Calendar ==Dye Coupling== *Following [[Daniel:Proto...")
 
>Djacobse
 
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===Labeling Results===
===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 15 uL
|style="font-weight:bold" width="71" | 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" | BSPP 1-546
| align="center" | 128.6
| align="center" | 4.7
| align="center" | 1.9
| align="center" | 3.0
| align="center" | 1.6
| align="center" | 1.2
| align="center" | 83
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | BSPP 2-594
| align="center" | 101.6
| align="center" | 4.8
| align="center" | 1.5
| align="center" | 2.4
| align="center" | 2.0
| align="center" | 1.6
| align="center" | 64
|}
Good results, especially BSPP 2.  Both of these should work well as controls, and I now have two different options/colors as negative controls.

Latest revision as of 21:46, 5 September 2013

BSPP Control (Started 09/03/2013)[edit]

Back to Calendar

Dye Coupling[edit]

0. Dry samples in a vaccuum centrifuge

For each

1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
2. Resuspend DNA in 5 uL nfH20
3. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer in PCR tube
4. Denature samples for 5 minutes at 95C, then snap cool using ice box
5. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
   *dye cannot be saved for later use.  Use immediately!
6. Add 2 uL dye to sample
7. Incubate in the dark for 1 hour
8. Add 10 uL nfH20 to sample
9. Purify using Centri Sep column
10. Measure concentrations using Microarray setting in nanodrop

Labeling Results[edit]

  ng/uL DNA pmol/uL dye ug DNA in 15 uL pmol/uL DNA dye/probe dye/100 bp Base:Dye Ratio (:1)
BSPP 1-546 128.6 4.7 1.9 3.0 1.6 1.2 83
BSPP 2-594 101.6 4.8 1.5 2.4 2.0 1.6 64

Good results, especially BSPP 2. Both of these should work well as controls, and I now have two different options/colors as negative controls.