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===Dye-coupling aminoallyl-dUTP=== Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-9-24 *Following [[Daniel:Protocols/AlexaDyeCouple | Dan's Dye Coupling Protocol]] and [[Daniel:Protocols/CentriSep | Dan's Column Size Separation Protocol]] *3 5ul samples: +aadUTP, -aadUTP, and NTC (just water) 1. Warm DMSO and sodium bicarbonate buffer to RT 2. Added 3ul of of sodium bicarbonate to 5ul sample 3. Denatured samples at 95C for 5 min and then snap cooled 4. Dissolved 1 vial of Alexa 564 dye in 6ul DMSO; vortexed and spun down 5. Added 2ul of dye to each sample 6. Incubated in dark for 1 hr 7. Added 15ul H2O to dye-coupled samples to make 25ul total 8. Took 0.5ul of dye-coupled sample and diluted to 10ul for '''non-filtered measurements''' 9. Added 24.5ul of sample to prepped Centri-Sep column and spun for 2 min at 750rcf 10. Measured dye conc and DNA conc using Nanodrop Microarray setting (dsDNA) {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Alexa 564 (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''DNA conc (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''DNA (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''Bases (pmole/ul)''' | align="center" style="background:#f0f0f0;"|'''Base:Dye Ratio''' |- | Filtered||+aadUTP||0.5||20.3||0.346074442||62.29339953||124.5867991 |- | Filtered||-aadUTP||0||25.5||0.434724053||78.25032945|| |- | Filtered||NTC||0||0.5||0.008524001||1.534320185|| |- | Nonfiltered||+aadUTP||17.7||14.7||0.25060563||45.10901345||2.548531833 |- | Nonfiltered||-aadUTP||16.3||13.9||0.236967229||42.65410115||2.616816022 |- | Nonfiltered||NTC||17.8||14.3||0.243786429||43.8815573||2.465256028 |} *90bp DNA mass: 58,658 g/mol *The Nonfiltered NTC is messed up, possibly could've taken the 0.5 ul from wrong tube in step 8 *Among the filtered samples, good evidence that the aadUTP is still good **Base:Dye ratio isn't the best but definitely falls within the range that Dan gets [[Daniel:ProbeList]] **He also uses 3x more dye for a reaction which could explain why I got less dye/base
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