I ordered a 40nmole synthesis scale, but I have received less than 40nmole product. Why is this?
The synthesis scale is related to the amount of the first base attached to the solid phase support, not the amount of final product. The amount of final product will be lower than the theoretical yield, and depends on the coupling efficiency and other losses during the transfer of material and quality control.
How do I prepare a 100μM solution of my oligo?
A Molar (1.0M) solution is 1 Mole of the oligo dissolved in 1 litre solution
A 100μM Solution is 100
μmoles/litre (or 100nmoles/ml)
Supposing the stated yield of the oligonucleotide is 309.9μg, and the molecular weight is 6319.
The Yield in nmoles is 309*1000/6319, or 49nmoles
To prepare a 100μmole solution 49 nmoles should therefore be dissolved in 490μl solution
How stable is my oligo?
The following table can be used as a guideline:
| State |
Temperature |
Stability |
| In solution |
-20°C |
>6 months |
| In solution |
Room Temperature |
<1week |
| Lyophilised |
-20°C |
>12 months |
| Lyophilised |
Room Temperature |
1-2 months |
When stored in solution, the solution should be buffered at pH7.0 and be free from nuclease contamination
What purification method should I request?
The method of purification depends, not only on the length of the oligo, but also on the application for which it is intended. The table below gives our suggested purifications for some of the more common applications:
| Application |
Suggested Purification |
| PCR |
RPC/Desalted |
| Cycle Sequencing |
RPC/Desalted |
| Fluorescent Sequencing |
HPLC |
| Cloning Experiments |
HPLC/Gel Purification |
| Mutagenesis |
HPLC/Gel/Purification |
| RT-PCR |
HPLC/Gel Purification |
| Antisense |
HPLC |
| Microarrays |
RPC/Desalted |
What is the maximum length of oligo I can order?
Because the coupling efficiency of the sequential reaction is never 100%, the percentage
of full-length product decreases with the length of the oligo. This decrease can be quite
dramatic, as shown in the following table:
|
% Yield of n-mer
|
| Base length |
99% Coupling |
98% Coupling |
| 20 Bases |
82 |
67 |
| 40 Bases |
67 |
45 |
| 60 Bases |
55 |
30 |
| 80 Bases |
45 |
20 |
| 100 Bases |
37 |
14 |
Because of this, and because adenine undergoes a low level of depurination during the
synthesis (which thus increases with time and the length of the oligo), we do not
recommend the synthesis of oligos greater than 100 bases in length. In all cases we
would recommend gel purification of any oligos longer than 60 bases.
What is the Tm listed on the Certificate of Analysis?
Tm is the symbol for Melting Temperature. This is the temperature at which 50% of a duplex
containing the sequence in question is single stranded. This is an important parameter for
gauging the annealing temperature to be used in a PCR reaction.
How do I calculate the Tm of my sequence?
We use the calculation:
Tm = 69.3 + GC%*0.41 – 650/(nA + nC + nG + nT)
Key in (or paste) your sequence below to find the estimated Tm
The inserted sequence of my clone appears to be different from the sequence ordered. What’s gone wrong?
The most likely explanations for apparent cloning errors are related to the chemistry used by all commercial manufacturers. These are as follows:
- The base G may be converted to the enol tautomer 2,6 diaminopurine, which is recognised as A by DNA polymerase. Clones containing an inserted oligonucleotide with this modified base will be perceived to have a G to A transition.
- The synthesis reactions may cause depurination, particularly affecting the base A. Depurinated oligos are usually degraded at the deprotection stage, but it is possible for a small percentage to remain. Clones containing an inserted oligo which is depurinated will appear to have an A or G deletion. Oligos containing a high purine content carry an increased risk of these artefacts.
- Failure sequences are capped to prevent them from taking any further part in the synthesis. Failure to cap such sequences will lead to them to continue synthesis, and clones containing such an oligo will contain deletions.
- A small percentage of incoming bases may couple with each other prior to coupling with the growing oligonucleotide chain. Clones containing an insert oligo of this kind will contain additions.
These events are extremely rare, but the risk of accumulating artefacts increases with exposure to the chemistry, and, therefore, to oligonucleotide length. We recommend the following steps to reduce these events interfering with your cloning experiments.
- Always use HPLC or Gel-purified oligos in cloning studies.
- Try to ligate sequences less than 35mers.
- Pick at least five clones.
- Always sequence across synthetic oligonucleotide inserts before proceeding to downstream applications.
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