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Adapter ligation to blunt ended plasmid

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#1 Rsm


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Posted 01 March 2012 - 01:29 AM

I am trying since some time to do an adapter ligation to a blunt ended plasmid (AmpR). The adapter is a PCR product (KanR gene plus my sequence), having an A overhang. I am treating my blunted plasmid with terminal deoxy transferase and ddTTP to have a single T for ligation. However, I can't get any colonies upon plating on Amp/Kan plates.

I am using ddTTP to avoid having a stretch of T's. However, isn't the hydroxyl group necessary for ligation? Can someone recommend a different approach for adator ligation to unknown target DNA? Thanks for any help!
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