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=== D: Primer Annealing and Template Switching Reaction === The DNA SMART Poly(dA) Primer anneals to the ssDNA template followed by DNA replication and template switching. At the end of this step, the DNA is double stranded with partial adapters on both ends. '''NOTE:''' Prepare the Template Switching Master Mix (Step V.D.5) prior to starting the primer annealing step below. :- Primer Annealing: Add 2 μl of the DNA SMART Poly(dA) Primer (yellow cap) to each reaction tube. Mix the contents of the tubes by gently vortexing and spin the tubes briefly." :- Incubate the tubes at 94°C in a preheated, hot-lid thermal cycler (ChIP-94) for exactly 1 min. :- Immediately remove the samples and place on ice for at least 2 minutes. :- Spin the tubes briefly to bring down any condensation. '''NOTE:''' Proceed immediately to the next step :- Prepare 6.2rxn Template Switching 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||6.00||37.20 |- | DNA SMART Oligonucleotide mix||6.00||37.20 |- | SMARTScribe Reverse Transcriptase||4.00||24.80 |- | Total||16.00|| |} '''Note:''' DO NOT add the SMARTScribe Reverse Transcriptase to the buffer until immediately prior to use. Mix well by gently vortexing and spin the tubes briefly in a micro centrifuge :- DNA Replication and Template Switching: Add the SMARTScribe Reverse Transcriptase (purple cap) to the Template Switching Master Mix prepared in Step V.D.5, then add 16 μl of the Template Switching Master Mix to each reaction tube. 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-D. Leave the tubes at 4°C until the next step :- '''ChIP-D:''' 42°C 90 min --> 70°C 15 min -- > 4°C hold '''*Note:''' I left the reaction O/N at 4C before continuing to PCR. <br>
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