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

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|style="font-weight:bold" | AmpR7
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| align="center" | 20
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| align="center" | 2.6
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Revision as of 21:17, 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