Higher ph of pcr buffers is another problem while ligase buffers have ph of around 7.5. However, one can reprecipitate pcr product and use in ligation. On Apr 13, 2015 10:35 PM, <methods-requ...@oat.bio.indiana.edu> wrote:
> Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-requ...@net.bio.net > > You can reach the person managing the list at > methods-ow...@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. T4 DNA ligase activity in PCR buffers (Bjorn) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sun, 12 Apr 2015 06:45:02 -0700 (PDT) > From: Bjorn <bjornj...@gmail.com> > Subject: T4 DNA ligase activity in PCR buffers > To: methods@net.bio.net > Message-ID: <f097451b-7bc1-42d6-b6cf-0963edc1c...@googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > 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 > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 114, Issue 2 > *************************************** > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
