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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
