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To add Illumina sequencing adaptors and custom indices, a second-round PCR was performed in a 40 l volume containing 2 l of the first-round product, 1 l each of 10 M P5 and P7 custom index primers, 0.4 l of Phusion High-Fidelity DNA Polymerase (NEB, no
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Most caregivers reading this article should start with sublingual methylcobalamin and confirm response with repeat lab testing in 8 to 12 weeks
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