Mass
Spectrometry
There is no single quality control
procedure that works perfectly on every oligonucleotide, which is why
our facility utilizes several approaches. However, one particularly useful
quality control tool for most oligonucleotide analyses is mass spectrometry.
This procedure is carried out at the Mass Spectrometry facility in the
UWBC run by Dr. Amy Harms.
The staff here at the Synthesis Facility has
been trained in the use of the on-site mass spec machine. We use MALDI-TOF
primarily, in which the oligonucleotide is mixed in a matrix, applied
to a plate, then subjected to a laser blast which ionizes the product
and sends it flying down a tunnel to a detector. The time of flight (TOF)
of the ionized product is inversely proportional to the molecular weight
of the oligonucleotide. Based on the TOF, the instrument assigns a molecular
weight to the various products detected following the laser blasts. Ideally
one peak of the correct MW is detected (the expected MW can be easily
determined using one of several on-line oligonucleotide calculators-see
the URL below for the one we typically use in our facility). Failure sequences,
if present, show up as a laddered array of peaks of descending MW, separated
by approximately 330 daltons (the MW of a base). In most instances, unless
a significant portion of the product resides in failure sequences, the
oligonucleotide will work fine for virtually any application.
A user can request to have their
oligonucleotide analyzed in this fashion for $5.00 (no free rides here,
all the facilities pay to use the other facilities). A hard copy of the
data is provided. You can request this on your order sheet by simply clicking
yes on the "Mass Spec Option" window. Keep in mind, though, that the oligo
will be ready before the data, so you'll have to come back for the hard
copy or wait to pick up the oligo.
We can also do this on oligonucleotides
from your freezer if you want to check the integrity of your stock. If
you're having problems on experiments utilizing oligonucleotides, it's
one way to possibly rule out that reagent as a problem. Call for more
details on what to provide, but typically we would need several ul of
a 30-50 ng/ul (5-10 pmol/ul for an 18-mer) solution. An important factor
to consider, however, is that very large oligonucleotides (>50 mers),
or particularly oligonculeotides with modifications, don't yield great
data on the mass spec. Results with such oligonucleotides may be ambiguous
and we can make no guarantees about the information gathered. Please call
for more information about this.