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=S2 Probe Prep ([[Daniel:Notebook/HiResChrPaint/2013-5-29|Started 5/29/2013]])= [[Daniel:Notebook/HiResChrPaint|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 [[Daniel:Notebook/HiResChrPaint/2013-6-10#Nanodrop Results|V6S2 set]] this afternoon. *Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|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 [[Daniel:Protocols/CentriSep|Centri-Sep Column]] ===Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="65" height="30" | |style="font-weight:bold" width="65" | ng/uL DNA |style="font-weight:bold" width="65" | pmol/uL dye |style="font-weight:bold" width="65" | ug DNA in 20 uL |style="font-weight:bold" width="71" | pmol/uL DNA |style="font-weight:bold" width="71" | dye/probe |style="font-weight:bold" width="71" | dye/100 bp |style="font-weight:bold" width="65" | Base:Dye Ratio (:1) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR9 | align="center" | 64.6 | align="center" | 0.6 | align="center" | 1.3 | align="center" | 3.9 | align="center" | 0.2 | align="center" | 0.3 | align="center" | 326 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR10 | align="center" | 71.1 | align="center" | 0.4 | align="center" | 1.4 | align="center" | 4.3 | align="center" | 0.1 | align="center" | 0.2 | align="center" | 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== {| 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" | |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" | |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" | |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" | |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" | |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" | |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" | |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 ==AmpR11 and AmpR12== 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=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="65" height="30" | |style="font-weight:bold" width="65" | ng/uL DNA |style="font-weight:bold" width="65" | pmol/uL dye |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR11 | align="center" | 32.3 | align="center" | 3.5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR12 | align="center" | 30.4 | align="center" | 5 |} Note that since is is pre-column it should be compared to [[Daniel:Notebook/HiResChrPaint/2013-7-10#Results|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.
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