2 replies to this topic

[MGDrzyzga@brandeis]

Does anyone know of good alternative buffers for use in PCR or strand displacement?  Currently I'm using the New England Biolabs thermopol buffer, which is Tris, Triton X-100, MgSO4, KCl, and (NH4)2SO4.  But the next step involves a reaction that's inhibited by Tris.  So I'm looking into alternative buffers.  I've tried desalting and transferring to a PBS buffer (with and without MgSO4), and it didn't work so well.  I know the second reaction (a CuAAC) is compatible with PBS.  So currently I'm left wondering if I can switch to a different buffer in the first place without disrupting the polymerase reaction.  I'm using BST polymerase to convert ssDNA to dsDNA while introducing an artificial nucleotide in place of dTTP.  It'd be nice if I could simply switch to a PBS buffer and not worry about desalting.

[phage434]

I'd recommend Bicine as your buffer.  Thermopol buffer has a pH of 8.8, so you want a buffer with a similar pKa.  Bicine has a pKa of 8.35, and has no free amine groups, which is the usual problem with Tris buffer incompatibility.  Bicine is a "Good" buffer, chosen for biological compatibility.  The phosphate in PBS is likely inhibitory to many reactions involving hydrolysis of dNTPs.  Tricine would be another (not as good) choice for a buffer.

[MGDrzyzga@brandeis]

Thanks.  I'll talk to my PI about it, but I'm concerned it might actually be just as bad.  Odds are that Tris inhibits the CuAAC ("click") reaction by binding to the cobalt ion.  Any tertiary amine is likely to cause similar problems.  But I wonder?...  The ligand usually used for the Cu is itself a tertiary amine, likely around the same pKa.  Maybe it could replace the Tris.  But that might be more complicated than it's worth, using a unique buffer.  Still, it's worth looking into whether bicine works.