Daniel:Notebook/HiResChrPaint/2013-7-11: Difference between revisions

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  trials (yes, it looks like I need more troubleshooting).  That will save on time and $$, and hopefully
  trials (yes, it looks like I need more troubleshooting).  That will save on time and $$, and hopefully
  I'll get to the bottom of this before long.
  I'll get to the bottom of this before long.
==Review of AmpR Experiments so Far==
{| 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="30"  valign="bottom" | &nbsp;
|style="font-weight:bold" width="65" align="center" | Sample
|style="font-weight:bold" width="65" align="center" | uL dye Added
|style="font-weight:bold" width="65" align="center" | Incubation Time (min)
|style="font-weight:bold" width="65" align="center" | pmol/uL DNA
|style="font-weight:bold" width="65" align="center" | pmol/uL dye
|style="font-weight:bold" width="65" align="center" | dye/probe
|style="font-weight:bold" width="65" align="center" | base:dye ratio
|- style="font-size:12pt"
| align="center" height="15"  valign="bottom" | &nbsp;
|style="font-weight:bold" align="center" | AmpR1
| align="center" align="center" | 1
| align="center" align="center" | 15
| align="center" align="center" | 3.5
| align="center" align="center" | 1.2
| align="center" align="center" | 0.341
| align="center" align="center" | 147
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | &nbsp;
|style="font-weight:bold" | AmpR2
| align="center" | 1
| align="center" | 15
| align="center" | 3.1
| align="center" | 1.2
| align="center" | 0.384
| align="center" | 130
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | X
|style="font-weight:bold" | AmpR3
| align="center" | 2
| align="center" | 15
| align="center" | 3.4
| align="center" | 1.9
| align="center" | 0.552
| align="center" | 91
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | *X
|style="font-weight:bold" | AmpR4
| align="center" | 5
| align="center" | 15
| align="center" | 1.9
| align="center" | 1.4
| align="center" | 0.7
| align="center" | 69
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | *
|style="font-weight:bold" | AmpR5
| align="center" | 2
| align="center" | 30
| align="center" | 3.3
| align="center" | 0.5
| align="center" | 0.2
| align="center" | 330
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | &nbsp;
|style="font-weight:bold" | AmpR6
| align="center" | 2
| align="center" | 60
| align="center" | 3.8
| align="center" | 0.4
| align="center" | 0.1
| align="center" | 474
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | X
|style="font-weight:bold" | AmpR7
| align="center" | 1
| align="center" | 20
| align="center" | 2.6
| align="center" | 2.3
| align="center" | 0.9
| align="center" | 56
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | *
|style="font-weight:bold" | AmpR8
| align="center" | 1
| align="center" | 20
| align="center" | 3.6
| align="center" | 0.7
| align="center" | 0.2
| align="center" | 259
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" height="15" | &nbsp;
|style="font-weight:bold" | AmpR9
| align="center" | 1
| align="center" | 15
| align="center" | 3.9
| align="center" | 0.6
| align="center" | 0.2
| align="center" | 326
|- style="font-size:12pt"
| align="center" height="15"  valign="bottom" | &nbsp;
|style="font-weight:bold" align="center" | AmpR10
| align="center" align="center" | 1
| align="center" align="center" | 20
| align="center" align="center" | 4.3
| align="center" align="center" | 0.4
| align="center" align="center" | 0.1
| align="center" align="center" | 539
|- style="font-size:12pt"
| align="center" height="15"  valign="bottom" | &nbsp;
|style="font-weight:bold" align="center" | BLANK
| align="center" align="center" | 0
| align="center" align="center" | 15
| align="center" align="center" | 4
| align="center" align="center" | 0.2
| align="center" align="center" | 0
| align="center" align="center" | 1009
|}
Notes: *denotes beginning of a new dye tube; X denotes the end

Revision as of 21:15, 11 July 2013

S2 Probe Prep (Started 5/29/2013)

Back to Calendar

ULYSIS Dye Coupling (Test 6)

Should be the last test.  I'm going to test at 15 minutes and 20 minutes incubation time, 1 uL each.  If the
results go well this morning I will be labeling the V6S2 set this afternoon. 
1. Add 1 uL DNA in 19 uL labeling buffer (Component C)
2. Denature DNA for 5 minutes at 95C; snap cool on ice
3. Add 1 uL dye to sample; add 4 uL labeling buffer
4. Incubate for 15 minutes (AmpR9) or 20 minutes (AmpR10) at 80C, snap cool on ice
5. Purify using Centri-Sep Column

Results

  ng/uL DNA pmol/uL dye ug DNA in 20 uL pmol/uL DNA dye/probe dye/100 bp Base:Dye Ratio (:1)
AmpR9 64.6 0.6 1.3 3.9 0.2 0.3 326
AmpR10 71.1 0.4 1.4 4.3 0.1 0.2 539
It would seem that I can always tell beforehand whether a yield will be good or bad, by the color of
the solution post incubation.  If the solution has a faint green color it will be a good yield.  If 
the solution remains clear it will be a bad yield.  Therefore I'm going to ignore columns for future 
trials (yes, it looks like I need more troubleshooting).  That will save on time and $$, and hopefully
I'll get to the bottom of this before long.

Review of AmpR Experiments so Far

  Sample uL dye Added Incubation Time (min) pmol/uL DNA pmol/uL dye dye/probe base:dye ratio
  AmpR1 1 15 3.5 1.2 0.341 147
  AmpR2 1 15 3.1 1.2 0.384 130
X AmpR3 2 15 3.4 1.9 0.552 91
*X AmpR4 5 15 1.9 1.4 0.7 69
* AmpR5 2 30 3.3 0.5 0.2 330
  AmpR6 2 60 3.8 0.4 0.1 474
X AmpR7 1 20 2.6 2.3 0.9 56
* AmpR8 1 20 3.6 0.7 0.2 259
  AmpR9 1 15 3.9 0.6 0.2 326
  AmpR10 1 20 4.3 0.4 0.1 539
  BLANK 0 15 4 0.2 0 1009

Notes: *denotes beginning of a new dye tube; X denotes the end