Oligonucleotides: I'm desperate
jrt at home.com
Sat Apr 1 21:18:58 EST 2000
A few suggestions. Not likely DNAse. The oligos are chemically
synthesized and purified without ever seeing protein. Could the
oligos be incompletely deprotected or insufficiently and variably
desalted? These might account for labeling problems and/or
mobility/band morphology problems. My experience is that PNK works
like a shot. You can calculate 1:1 molar ratio of NTP to free ends
and get nearly 100% incorporation. So if you add excess NTP (say 2X)
and quantitate the incorporation you can pretty well assume the degree
of incorporation defines the true concentration of free 5' hydroxyls.
If that doesn't jive with the UV data, question your oligo supplier
re: quality. You might try the same quantitation experiment with some
dephosphorylated plasmid DNA just to convince yourself the
quantitation is accurate.
To quantitate the incorporation conveniently by paper chromatography
Spot the samples on whatman 3MM and develop in 3.5 M Ammonium sulfate,
75 mM Phosphate pH 6.8 with 2% n-Propanol (not isopropanol; the
n-propanol isn't miscible but forms a vapor barrier to limit
evaporation; I actually omit the n-propanol and just use fresh
solution each time instead). NTPs move up the paper (Rf ~0.5-0.8);
Oligos remain at the origin. Slap a film on to find the spots and cut
and count to quantitate precisely. Unfortunately, I've lost the
journal ref for this.
Merck Research Labs
January Weiner <nospam_jweiner1 at ix.urz.uni-heidelberg.de> wrote:
> I have a problem. A reallly big problem -- for me. I have ~40
> oligonucleotides which are supposed to be used for quantitative
> Northern Blotting. I have quantified them measuring OD at 260 nm,
> three times each -- trying to be very exact. I have labelled them
> with PNK and gamma-32P-ATP. I have looked at them on a PAGE.
> And I get very different results for all oligonucleotides. For
> example, I have two oligonucleotides which are both 18mers and have
> the same OD, look totally different on a PAGE (one is barely seen,
> the other has a very thick band), and yield different CPMC/ul
> (although this does not correlate with the thickness of the PAGE
> band, and obviously does not correlate with the assumed oligo
> concentration, for I tried to label 5 pmol in each case).
> I've been stuck with this problem already for weeks. Repeated
> everything. Twice. Three times. One idea was, DNAse activity -- but
> in TE? And besides, there are no traces over that time, and all the
> oligos were newly synthetized.
> Any suggestions? (or maybe I should start working as a taxi
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