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=== B: Dephosphorylation of 3’ Ends === :- Prepare 6.2 rxn Dephosphorylation Master Mix for all reactions by combining the following reagents on ice. ::{| {{table}} | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:100px;background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''6.2 rxn''' |- | DNA SMART Buffer||3.25||20.15 |- | Shrimp Alkaline Phosphatase ||0.75||4.65 |- | Total||4.00|| |} '''NOTE:''' Add the Shrimp Alkaline Phosphatase to the buffer immediately prior to use. Mix well by gently vortexing and spin the tubes briefly in a microcentrifuge. Keep the DNA SMART Buffer on ice until its next use in Step V.D. :- Add 4 μl of the Dephosphorylation Master Mix to each reaction tube from Step V.A. Mix the contents of the tubes by gently vortexing, and spin the tubes briefly" :- Place the tubes in a preheated thermal cycler and run program ChIP-B. Leave the tubes at 4°C until the next step<br> :- '''ChIP-B:''' 37°C 10 min --> 65°C 5 min --> 4°C hold<br> '''NOTE:''' During the 65°C incubation step, prepare the T-Tailing Master Mix in Step V.C.1 below. <br>
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