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DNA subcloning question
Posted by: ludeer (IP Hidden, New member, 1)
Date: July 6, 2005 07:36PM

hi,

I am doing sticky ends subcloning into plasmid vector. I CIAP treated both my DNA insert and linerized plasmid, did the gel purification and stored them at -80C freezer for over the weekend. After transformation I only got very few colonies. Is it a bad practice to freeze sticky ends DNA for long time? I am afraid of something chewing off the ends. Another question: is it necessary to ethanol purify DNA followed by phenol:chloroform (1:1) extraction? will it give higher ligation efficiency? I would appreciate any suggestions or references.

Thank you!

 

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