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Daniel:Notebook/HiResChrPaint/2013-7-9
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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 3)== Based on [[Daniel:Notebook/HiResChrPaint/2013-7-8|yesterday's results]] I think I'm going to see what happens if I use all 5 uL of dye available (see ULYSIS protocol) in one labeling. I'll use the oligo Noi gave me again, as I have a lot and it doesn't waste probe. *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 5 uL dye to sample 5. Incubate for 15 minutes at 80C, snap cool on ice 6. 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="65" | pmol/uL DNA |style="font-weight:bold" width="65" | dye/probe |style="font-weight:bold" width="65" | 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" | AmpR4 | align="center" | 31.8 | align="center" | 1.4 | align="center" | 0.636 | align="center" | 1.9 | align="center" | 0.7 | align="center" | 1.45 | align="center" | 69 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | BLANK | align="center" | 66.6 | align="center" | 0.2 | align="center" | 1.332 | align="center" | 4.0 | align="center" | 0.0 | align="center" | 0.10 | align="center" | 1009 |} This is not far off from the suggested efficiency values (44-67:1). I think that the efficiency can be improved, however, by also incubating longer. ==ULYSIS Dye Coupling (Test 4)== Based on result from above, I think that using more dye is helpful. However, I also want to see if I can get the same result using less dye and more time. *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) for AmpR5 and AmpR6; 3. Denature DNA for 5 minutes at 95C; snap cool on ice 4. Add 2 uL dye to sample; add 3 uL labeling buffer 5. Incubate AmpR5 for 30 minutes at 80C; incubate AmpR6 for 1 hour, snap cool on ice 6. Purify using [[Daniel:Protocols/CentriSep|Centri-Sep Column]] {| 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="150" 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" | AmpR5 (30 min incubation) | align="center" | 54.5 | align="center" | 0.5 | align="center" | 1.1 | align="center" | 3.3 | align="center" | 0.2 | align="center" | 0.3 | align="center" | 330.3 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | AmpR6 (1 hr incubation) | align="center" | 62.6 | align="center" | 0.4 | align="center" | 1.3 | align="center" | 3.8 | align="center" | 0.1 | align="center" | 0.2 | align="center" | 474.2 |} I'm suspicious of these results. It's possible that the extra time completely erradicated the dyes, but I find it more likely that there was an error in the process. Post-incubation, the samples have had a faint green color that grows stronger with increasing concentration. This experiment neither showed any hint of a green color, and the pmol/uL values are low enough that I am suspicious they are erroneous.
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