|
1. What
is the minimum and maximum oligo length required?
The
minimum length of an oligonucleotide is 4 bases; the maximum
length of oligonucleotides offered by TCGA is 100 bases for
and above 50 nmole scale sythesis .
2.Why is it necessary to purify oligonucleotides?
Impurities
which occur during the oligonucleotide synthesis have to be
removed subsequently:
- It is a principle of chemistry that a chemical reaction
never processes completely. The main side reaction, with the
use of phosphoramidite chemistry, is the formation of n-x
products. The longer the sequence, the more n-x products
occur.
- After deprotection, the protecting groups will be part of
the raw synthesis product.
- Due to the large amounts of solvents and the inorganic
materials used, salt traces in the raw product are
unavoidable.
Therefore purification of oligonucleotides is necessary to
avoid potential impairments in many applications through the
final oligonucleotide product.
3. What
quality control measures are used at TCGA?
TCGA we
use MALDI-TOF - (Matrix Assisted Laser
Desorption Ionization – Time of
Flight) mass spectrometers or PAGE (Polyacrylamide
Gel Electrophoresis) to perform routine
check of oligonucleotides.
4. Why is the yield sometimes not reproducible?
Obviously,
many factors influence the yield. The main reason why you
might see different yields for the same oligo is due to the
column loading with CPG (controlled pore glass) material
used to initiate the synthesis. The minimum loading
corresponds to the synthesis scale but fluctuation of the
CPG amount and CPG loading can occur which will lead to
varying yields.
5. How
long are unmodified oligonucleotides stable and under which
conditions should I store them?
We usually
supply unmodified oligonucleotides in lyophilized state,
since this form is less sensitive to degradation by
nucleases and more stable for transportation. We recommend
you to store this lyophilized state at a temperature of -20
°C or below.
Once you have dissolved your oligonucleotides in sterile
water or buffer solution, the best way to keep them is to
aliquot them into several tubes, lyophilize the aliquots,
and store them at -20 °C. The sample you are using can be
kept in a refrigerator at 4° C for a short time to avoid
continuous freezing and thawing of the solution.
6. How
should fluorescent oligonucleotides be stored?
The best
way to store them is to aliquot the oligo into several
tubes, and store the aliquots at -20°C. The sample you are
using can be kept in a refrigerator at 4°C for a short time
to avoid continuous freezing and thawing of the solution.
Additionally, we strongly recommend to keep dye labled
oligonucleotides out of light at any case.
7. How
should the oligos be resuspended?
For long-term storage we recommend that the oligos be
dissolved in a buffer, such as TE (10 mM Tri-HCl, 1 mM EDTA,
pH 8.0), instead of just sterilized water. Once
resuspended, oligos should be kept frozen at -20°C for
longer use.
8. If oligos were left at room temperature for more than a week,would they still work?
Once dried, oligos are supposed to have a tremendous
stability. Even in solution, they are reasonably stable.
Therefore, for the most of cases, without the contamination
of materials which can cause decomposition of oligos, they
should still work well, even if they were left at room
temperature for more than a week
9.How
do I order a wobble/degenerated base?
A mixture
of bases is commonly known as wobble or degenerated base.
Please find the universal nomenclature in the following
table.
When
ordering a wobble, equal amounts of different
phosphoramidites are used in one coupling reaction during
synthesis. Not all nucleosides are equally incorporated
during synthesis, you might see a slight bias at the wobble
position in the final product.
|
