Daniel:Notebook/HiResChrPaint/2014-1-15: Difference between revisions

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>Djacobse
>Djacobse
Line 11: Line 11:
  3. [[Daniel:Protocols/CentriSep|Centri-Sep column purification]]
  3. [[Daniel:Protocols/CentriSep|Centri-Sep column purification]]
  4. Measure in nanodrop
  4. Measure in nanodrop
===Nanodrop Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt"
| align="center" width="65" height="15"  valign="bottom" | &nbsp;
|style="background-color:#8DB4E2;font-weight:bold" width="65" colspan="4" align="center" valign="bottom" | Pre Column
|style="background-color:#E6B8B7;font-weight:bold" width="65" colspan="4" align="center" valign="bottom" | Post Column
|- style="font-size:12pt;font-weight:bold" align="center"
|style="background-color:#D9D9D9" height="30" | Sample
|style="background-color:#8DB4E2" | ng/uL dsDNA
|style="background-color:#8DB4E2" | pmol/uL dye
|style="background-color:#8DB4E2" | pmol/uL ss probe
|style="background-color:#8DB4E2" | dye/100bp
|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" | dye/100bp
|- style="font-size:12pt" align="center"
|style="background-color:#D9D9D9;font-weight:bold" height="15" | Alu2D
|style="background-color:#8DB4E2" align="center" | 9.2
|style="background-color:#8DB4E2" align="center" | 0.3
|style="background-color:#8DB4E2" align="center" | 0.3
|style="background-color:#8DB4E2" align="center" | 1.1
|style="background-color:#E6B8B7" align="center" | 8.3
|style="background-color:#E6B8B7" align="center" | 0.1
|style="background-color:#E6B8B7" align="center" | 0.3
|style="background-color:#E6B8B7" align="center" | 0.4
|- style="font-size:12pt" align="center"
|style="background-color:#D9D9D9;font-weight:bold" height="15" | Blank
|style="background-color:#8DB4E2" align="center" | 0
|style="background-color:#8DB4E2" align="center" | 0
|style="background-color:#8DB4E2" align="center" | 0.0
|style="background-color:#8DB4E2" align="center" | 0.0
|style="background-color:#E6B8B7" align="center" | 0.4
|style="background-color:#E6B8B7" align="center" | 0
|style="background-color:#E6B8B7" align="center" | 0.0
|style="background-color:#E6B8B7" align="center" | 0.0
|- style="font-size:12pt" align="center"
|style="background-color:#D9D9D9;font-weight:bold" height="15" | Cot1
|style="background-color:#8DB4E2" align="center" | 10.3
|style="background-color:#8DB4E2" align="center" | 1.3
|style="background-color:#8DB4E2" align="center" | 0.2
|style="background-color:#8DB4E2" align="center" | 4.2
|style="background-color:#E6B8B7" align="center" | 10.7
|style="background-color:#E6B8B7" align="center" | 0.9
|style="background-color:#E6B8B7" align="center" | 0.2
|style="background-color:#E6B8B7" align="center" | 2.8
|- style="font-size:12pt" align="center"
|style="background-color:#D9D9D9;font-weight:bold" height="15" | V6S3
|style="background-color:#8DB4E2" align="center" | 17.6
|style="background-color:#8DB4E2" align="center" | 0.5
|style="background-color:#8DB4E2" align="center" | 0.7
|style="background-color:#8DB4E2" align="center" | 0.9
|style="background-color:#E6B8B7" align="center" | 17.8
|style="background-color:#E6B8B7" align="center" | 0.2
|style="background-color:#E6B8B7" align="center" | 0.7
|style="background-color:#E6B8B7" align="center" | 0.4
|}

Revision as of 22:53, 15 January 2014

Alu Control (Started 1/08/2014)

Back to Calendar

Dye Coupling Test (Continued from 1/14/14)

The Cot1 data has been routinely coming back with spurious results, showing greater amounts of DNA than either the Alu sample or the V6S3 control. Since this result makes no sense, I'm going to do a little testing today to see if I can find the problem. The easiest thing to do is to run all 4 samples on columns again. If the DNA was blocking the dye, then once again more dye should stay in the column, and the Cot1 sample should drop to a near-zero dye value while still retaining DNA. However, since the Alu and V6S3 samples should be covalently linked to fluorophores, the relative dye ratio should stay pretty consistent from before the test to after the test. Blank should be zero both before and after.

1. Add 5 uL nfH2O to each sample
2. Measure in nanodrop
3. Centri-Sep column purification
4. Measure in nanodrop

Nanodrop Results

  Pre Column Post Column
Sample ng/uL dsDNA pmol/uL dye pmol/uL ss probe dye/100bp ng/uL ssDNA pmol/uL dye pmol/uL ss probe dye/100bp
Alu2D 9.2 0.3 0.3 1.1 8.3 0.1 0.3 0.4
Blank 0 0 0.0 0.0 0.4 0 0.0 0.0
Cot1 10.3 1.3 0.2 4.2 10.7 0.9 0.2 2.8
V6S3 17.6 0.5 0.7 0.9 17.8 0.2 0.7 0.4