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=Dye coupling SNAP25 odd&even 488&594= ==Combine SNAP25_odd and SNAP25_even== *Combined SNAP25 odd probes (24) and even probes (24) following same protocol as [[Matt:LabNotes/2015-7-8#Combining_CUX2_odd_and_CUX2_even | CUX2 odd&even]] **SNAP25_odd: 1148.3 ng/ul **SNAP25_even: 1154.0 ng/ul ==Dye coupling== [[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> [[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] *SNAP25_odd-488 *SNAP25_even-594 #Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature #Add 7.5 uL DNA (~1ug/ul) and 4.5 uL of sodium bicarbonate buffer #Denature samples for 5 minutes at 95C, then snap cool using ice box #Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing #*Dye cannot be saved for later use. Use immediately! #Add 8 uL sample to dye tube (Mixed 12ul but after heating only ~8ul left) #Incubate in the dark for 1 hour #Add 10 uL 3M NaOAc and 80 uL '''H2O''' to sample #*Previously did 80ul TE but maybe H2O will work better because no amine groups #[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation #*Use 1X TE Buffer #*No vacufuge #Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight #Centrifuge at 4 C for 30 minutes (14,000 rpm) #Remove supernatant and add 750 uL chilled 75% EtOH #Centrifuge 5 minutes at 4 C #Dry the pellet in the hood #Resuspend pellet in 12 uL TE #Check dye:probe ratio with Nanodrop #*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) #*Starting DNA mass = 7.5ul * 1ug/ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe''' | align="center" style="background:#f0f0f0;"|'''ssDNA (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''594 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''dye:probe ratio''' | align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' |- | SNAP25_odd-488||413.9||66.1||-0.1||1.04||5.0||7.5||66.667 |- | SNAP25_even-594||455.3||-1.2||89||1.27||5.5||7.5||73.333 |- | SNAP25_even-594||477.3||-1.4||88.3||1.20||5.7||7.5||76 |}
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