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=S2 Probe Prep ([[Daniel:Notebook/HiResChrPaint/2013-5-29|Started 5/29/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Summary of ULYSIS Tests using AmpR Oligo== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="30" | Sample | width="65" | uL dye Added | width="65" | Incubation Time (min) | width="65" | pmol/uL DNA | width="65" | pmol/uL dye | width="65" | dye/probe | width="65" | base:dye ratio |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR1 | align="center" | 1 | align="center" | 15 | align="center" | 3.5 | align="center" | 1.2 | align="center" | 0.341 | align="center" | 147 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | 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" |style="font-weight:bold" height="15" | 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" |style="font-weight:bold" height="15" | 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" |style="font-weight:bold" height="15" | 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" |style="font-weight:bold" height="15" | 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" |style="font-weight:bold" height="15" | BLANK | align="center" | 0 | align="center" | 15 | align="center" | 4 | align="center" | 0.2 | align="center" | 0 | align="center" | 1009 |} ==ULYSIS Dye Coupling (Test 5)== Based on [[Daniel:Notebook/HiResChrPaint/2013-7-9#ULYSIS Dye Coupling (Test 4)|yesterday's results]] I want to retry the time experiment, this time only with 20 minutes instead of 30 or 60. Also, I'm going to use 1 sample of the dye I made yesterday (AmpR7) and one with a "fresh" dye I'll prepare today (AmpR8). Hopefully this will explain the weird results from yesterday. Also, I'm going to do a pre/post comparison for the column, to see what that yields. *Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS protocol]] 1. Add 5 uL DMSO to Alexa dye 488 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 20 minutes at 80C, snap cool on ice 6. Measure concentrations in Nanodrop 7. Purify using [[Daniel:Protocols/CentriSep|Centri-Sep Column]] ===Results=== '''Labeling Efficiency''' {| 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="71" | Base:Dye Ratio (:1) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR7 | align="center" | 42.3 | align="center" | 2.3 | align="center" | 0.8 | align="center" | 2.6 | align="center" | 0.9 | align="center" | 1.8 | align="center" | 56 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR8 | align="center" | 59.9 | align="center" | 0.7 | align="center" | 1.2 | align="center" | 3.6 | align="center" | 0.2 | align="center" | 0.4 | align="center" | 259 |} '''Column Efficiency''' {| 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="45" | |style="font-weight:bold" width="72" | Pre ng/uL DNA |style="font-weight:bold" width="67" | Pre pmol/uL dye |style="font-weight:bold" width="65" | ng/uL DNA |style="font-weight:bold" width="65" | pmol/uL dye |style="font-weight:bold" width="78" | Column Yield (% DNA) |style="font-weight:bold" width="80" | Column Yield (% dye) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR7 | align="center" | 61.4 | align="center" | 15.4 | align="center" | 42.3 | align="center" | 2.3 | align="center" | 59.9 | align="center" | 13.0 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR8 | align="center" | 60.8 | align="center" | 2.8 | align="center" | 59.9 | align="center" | 0.7 | align="center" | 85.7 | align="center" | 21.7 |} Not really sure what happened here. First I'll note that I couldn't get a full uL of dye from the old tube, so I actually got better results than ever from <1 uL dye. The results would suggest I mixed up the tubes, since I would have expected the dye made today to perform better.
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