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

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>Djacobse
(Created page with "=Alu Control (Started 1/08/2014)= Back to Calendar ==Dye Coupling Test ([[Editing Daniel:Noteboo...")
 
>Djacobse
 
(3 intermediate revisions by the same user not shown)
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[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]


==Dye Coupling Test ([[Editing Daniel:Notebook/HiResChrPaint/2014-1-14|Continued from 1/14/14]])==
==Dye Coupling Test ([[Daniel:Notebook/HiResChrPaint/2014-1-14|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.
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.
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  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"
| width="65" height="15"  valign="bottom" | 
|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
| align="center" width="65"  valign="bottom" | &nbsp;
|- 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="background-color:#D8E4BC" | dye/100bp retention (%)
|- 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="background-color:#D8E4BC" align="center" | 36.9
|- 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="background-color:#D8E4BC" | NA
|- 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="background-color:#D8E4BC" align="center" | 66.6
|- 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
|style="background-color:#D8E4BC" align="center" | 39.6
|}

Latest revision as of 23:09, 15 January 2014

Alu Control (Started 1/08/2014)[edit]

Back to Calendar

Dye Coupling Test (Continued from 1/14/14)[edit]

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[edit]

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 dye/100bp retention (%)
Alu2D 9.2 0.3 0.3 1.1 8.3 0.1 0.3 0.4 36.9
Blank 0 0 0.0 0.0 0.4 0 0.0 0.0 NA
Cot1 10.3 1.3 0.2 4.2 10.7 0.9 0.2 2.8 66.6
V6S3 17.6 0.5 0.7 0.9 17.8 0.2 0.7 0.4 39.6