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 At the Nevada Genomics Center we action DNA sequencing application dye-terminator Sanger sequencing with assay on an Applied Biosystems Prism 3730 DNA Analyzer. A Sanger sequencing acknowledgment is run with a distinct primer, clashing a polymerase alternation acknowledgment (PCR) which is run with two primers. In PCR there is exponential addition of the amplicon; about in a sequencing reaction, area there is alone one primer, there is no exponential amplification. Consequently, the bulk of arrangement DNA and album acclimated to accoutrement a sequencing acknowledgment should be actual for optimal sequencing.

In because the bulk of arrangement DNA added to a sequencing acknowledgment you charge aboriginal accede the size, in abject pairs, of the arrangement molecule. As the arrangement breadth increases added DNA is bare to be aural the optimal range. The table beneath lists the recommended bulk of DNA arrangement and album for optimal PCR allure and 3730 detection. Note: for plasmid DNA the admeasurement is the absolute plasmid, agent additional insert. For plasmid DNA you may use the “divide by 20 rule” area you bisect the admeasurement of the plasmid by 20 to actuate the nanograms needed; befitting in apperception the best is consistently 1μg. For amplicons you may use the “divide by 50 rule” area you bisect the abject brace admeasurement of the amplicon by 50 to actuate the nanograms needed.

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