PCR ghost bands
lwashing at sunflower.bio.indiana.edu
Tue Jul 5 12:44:28 EST 1994
In article <1994Jul2.162223.1036 at hydra.acs.ttu.edu>, hepad at ttacs.ttu.edu
(Peter Doris) wrote:
> Does anyone care to recommend a column and method for HPLC purification
> of primers. Since I imagine many such methods work in anion exchange,
> what deslating steps for HPLC purified primers do people prefer.
> Further, how much primer do you run so as not to overload the column?
Ion exchange HPLC is a great way to purify oligonucleotides if you have the
equipment. Fast, no organic solvents needed, no acrylamide gels, etc.
We have been using Pharmacia's Mono Q for many years and more recently
Resource Q strong anion exchange columns. They separate fragments well if
the oligo is less than 30 or so bases long. Binding capacity is huge; we
load the whole batch if we want to. They are pricey but will last for
years if properly cared for. Other companies also make strong ion exchange
columns which might be just as good.
Buffers are at pH12, and the eluent buffer is 1M NaCl.
We desalt on little disposable reverse phase C18 columns (eg., Waters's
Sep-Pak cartridges or Analytichem's Bond Elut or many others.)
(Now, has anyone compared Mono Q with other anion exchange columns for
oligos?? A couple years ago we bought a Millipore QMA MemSep, which they
were touting as giving Mono-Q performance, and we were rather dissappointed
in the results. There must be others.)
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