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The second adapter called the "common" adapter was ligated to the overhang end of NlaIII.
A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP).
The iDAPT i4 is available for $59.99 from the company website, and comes with 6 of the most common adapter tips; microUSB, miniUSB, iPod/iPhone, Samsung, Nokia, and Sony Ericsson.
The second, or "common", adapter has only an ApeKI-compatible sticky end: 5'-CWGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG and 5'-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT (Figure 1).
Fragments under 64 base pairs result in the presence of either the common adapter or an internal ApeKI recognition sequence (from partial digestion or chimera formation) within 64 bases of the end of the barcode.
Recent work has shown that the IκB kinase (IKK γ scaffolding protein is the final common adapter protein required by RIG-I·MAVS to activate divergent rate-limiting kinases downstream controlling the NF-κB and IRF3 pathways.
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The Symmetrix and Clariion lines will also use common adapters.
Barcode and common adapters were then quantified using an intercalating dye (PicoGreen®; Invitrogen, Carlsbad, CA), diluted in water to 0.6 ng/µL (∼02 pmol/µL), mixed together in a 1∶1 ratio, and 6 µL (∼0.06 pmol each adapter) of the mix was aliquoted into a 96-well PCR plate and dried down.
Barcode sequence, common adapters and PCR primers used.
For libraries 1 3, 48 barcode plus common adapters were used (Elshire et al. 2011).
PCR will amplify only those restriction digested DNA fragments that have barcode adapters added on one end and common adapters added on the other end.
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