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do 5'-phosphorylated primers expire?

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do 5'-phosphorylated primers expire?

Postby vastgenome » Tue May 04, 2010 2:09 am

Hi guys,

A naive question, as i m trying to introduce specific point mutations in the middle of a gene linked in a plasmid. The way i do it is through PCR off the entire plasmid and then reclose the circle. I ran into some problems ligating the linear PCR product, and i was wondering if the modified primers (5'-phosphorylated) could lose the 5' phosphate through frequent thawing/freezing cycles.

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Postby jonmoulton » Tue May 04, 2010 3:38 pm

Freeze-thaw won't break off a covalent bond on a monophosphate, but enzymatic activity could.
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