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Old 07-06-2011, 06:35 AM   #7
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Location: Purdue University, West Lafayette, Indiana

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Originally Posted by ETHANol View Post
Thanks. I put the order in. We'll see if it works.

The phosphorotiolation is suppose to increase efficiency with proofreading polymerases. Maybe it's not necessary but it couldn't hurt.
Just to be clear:
The phosphorothioate linkages are not susceptible to (or are less susceptible to?) exo-nucleolytic enzymes.

The hallmark of a high fidelity enzyme is a 3'->5' exonuclease activity. So there is a connection there. But really the idea is that if your adapters have 3' overhangs that you don't want to get nibbled back by "end polishing" enzymes (eg T4 polymerase) that did not get completely removed/deactivated in an earlier step, then you put in the phosphorothioate linkages.

pmiguel is offline   Reply With Quote