Oligonucleotide concentration
Peter Myler
mylerpj at u.washington.edu
Fri Feb 10 18:07:50 EST 1995
Some months ago there was some discussion about how to calculate oligo
MW. I have been doing some research on the subject and have found it
rather confusing. Perhaps some-one out there can comment on the
following findings.
As far as I can tell the most comprehensive formula for calculating MW
is (for oligodeoxyridonucleotides):
MW = [(251 x nA) + (245 x nT) + (267 x nG) + (230 x nC) + (61 x n-1) +
(54 x n) + 17 x n-1) + 2]
This formula (from "Oligonucleotides and Analogs: A practical
Approach by Eckstein, IRL Press) accounts for each base, the phosphate
group between them (but not at 5' end), three water molecules per base
and the ammonium cation for each phosphate group. Note that the atomic
weights used are only approximations.
I have also seen a number of other formulas, most of which do not account
for the water and ammonium ions. lso note that different computer
programs use different formulae, as do different oligo suppliers.
Actually none of this makes any difference (I think) if you express
everything in uM, rather than ug/ml, since the concentrations are
determined from the absorbance and molar extinction co-efficients.
However, this raises more questions: what formula to use and what
wavelength to use.
The same source as above suggests using:
E254nm = [(8.8 x nT)+(7.3 x nC)+(11.7 x nG)+(15.4 x nA)]x 0.9
The factor of 0.9 is to correct for the effect of base stacking, which
lessens the absorbance.
However, I have also seen values of E260 of 8.7 (T), 7.4 (C), 11.5 (G)
and 15.4 (A) from Handbook of Biochemistry and Molecular Biology (1975)
Fasman, G.D. CRC Press.
So, any comments on what one is to do!!!!
===============================================================================
Peter J. Myler phone: (206) 284-8846x332
Seattle Biomedical Research Institute FAX: (206) 284-0313
4 Nickerson Street e-mail: MYLERPJ at U.WASHINGTON.EDU
Seattle, WA 98109-1651
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