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T4 DNA ligase activity in PCR buffers

Bjorn via methods%40net.bio.net (by bjornjobb from gmail.com)
Sun Apr 12 08:45:02 EST 2015


Hi all,

We routinely make blunt end ligations with unpurified PCR products in the lab.
We sometimes see reduced efficiency when a large portion of the ligation mixture is unpurified PCR product. I was wondering if the PCR buffers might have a negative effect on T4 DNA ligase activity?


This pdf from metabion (http://www.metabion.com/downloads/datasheets/enzymes/ligase/mi-E0149.pdf) says that T4 DNA ligase is severely inhibited by KCl and NaCl above 200mM. That information is probably from here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC341233/

Some standard recipes contain much more KCl. This one from csh has 500mM (http://cshprotocols.cshlp.org/content/2006/1/pdb.rec463.full?text_only=true)

We normally use a quite strongly buffered recipe from Fermentas:  

FERMENTAS 10X Taq Buffer with (NH4)2SO4
750 mM Tris-HCl (pH 8.8 at 25°C)
200 mM (NH4)2SO4
0.1% (v/v) Tween 20.

This buffer has no KCl, but a lot of Tris and ammonium sulphate. Is there any available information on ammonium sulfate inhibition?

Thanks for input

/bjorn 




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