Daniel:Notebook/HiResChrPaint/2013-7-11

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Revision as of 22:44, 11 July 2013 by >Djacobse
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S2 Probe Prep (Started 5/29/2013)[edit]

Back to Calendar

ULYSIS Dye Coupling (Test 6)[edit]

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[edit]

  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[edit]

  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

AmpR11 and AmpR12[edit]

Trying again to make sense of this dye procedure.  The above table suggests that the last few drops of
the dye per reaction tube always seem to be better than the first.  It might just be a problem of mixing.
So I'm going to try again but this time mix it really well (vortex and pipette) before I add the dye.
1. Add 5 uL DMSO to vial of dye; vortex vigorously and pipette thoroughly.
2. Add 1 uL DNA in 19 uL labeling buffer (Component C)
3. Denature DNA for 5 minutes at 95C; snap cool on ice
4. Add 1 uL dye to sample; add 4 uL labeling buffer
5. Incubate for 15 minutes at 80C, snap cool on ice

AmpR11 and AmpR12 Results[edit]

  ng/uL DNA pmol/uL dye
AmpR11 32.3 3.5
AmpR12 30.4 5
Note that since is is pre-column it should be compared to yesterday's results.  The numbers are slightly better
than AmpR8 (which didn't work) but nowhere near as good as AmpR7 (which did work), and the solution wasn't
green leading me to believe it still isn't working well.