Daniel:Notebook/HiResChrPaint/2013-7-11: Difference between revisions
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Revision as of 21:17, 11 July 2013
S2 Probe Prep (Started 5/29/2013)
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.
- Following ULYSIS protocol
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