Frequently Asked Questions (FAQ)
I am a new customer, how do I order?
Ordering guide for our Webshop:
- First time user, choose: “New customer”
- Fill information in and press enter
- Wait for reply e-mail with log in information. Your information needs to be approved, which will be done as quickly as possible.
- When approved, you can log into the Webshop with: username and password
- You can edit user details. Your account is based on your e-mail address.
How is the Tm of an oligonucleotide calculated?
The Tm (the temperature at which 50% of the oligonucleotide-duplex is melted) is of critical importance when designing oligos. Typically, annealing or hybridizations are performed at 5-10⁰C below the Tm of the duplex. In addition, the difference in Tm of a primer pair (e.g., forward and reverse primer) should be below 4⁰C for successful PCR.
Melting point calculations in Webshop:
For oligonucleotides < 15 bases the Tm is determined by the Marmur formula:
For oligos <15 bases: Tm= (wA+xT) * 2 + (yG+zC) * 4
However for longer oligos, the simple Marmur formula doesn’t take into account the increasing contribution of factors such as oligonucleotide concentration, salt concentration and oligonucleotide sequence, in the Tm equation. For oligonucleotides above 15 bases the following formula is generally considered to provide an accurate prediction of Tm
For oligos 15-70 bases: Tm = (ΔH⁰)/(ΔS^0+ R*Ln(C1-(C2/2)))- 273,15⁰C
A concentration of 50mM Na+ is the base for this calculation.
We do not provide Tm calculations for oligos above 70 bases, since the complexity of variables as well as the formation of secondary structures in longer products, prevents accurate Tm predictions. The above mentioned formulas do not take into consideration Tm changes induced by modifications.
What are "wobbles"?
When comparing multiple sequences, one may find that alignment reveals no region with sufficient consensus to accommodate a unique single oligonucleotide for use as a primer or probe. In some cases, only one or two nucleotides are mismatched. When designing primers for those regions, one may choose to introduce a degenerate site, or "wobble", to compensate for the variability in the target sequence. Letter codes are used to represent the combination of two or more different nucleotide phosphoramidites blended at equimolar ratios prior to coupling at that position in the sequence. The final product is a blend of two or more different sequences made simultaneously during one synthesis.
2 nucleotide wobble
R = A+G
W = A+T
M = A+C
Y = C+T
S = C+G
K = G+T
3 nucleotide wobble
B = C+T+G
D = A+G+T
H = A+C+T
V = A+C+G
N = A+C+T+G
Why does the synthesis scale ordered for my oligonucleotide not correspond with the final yield?
The synthesis scale represents the quantity of precursor used to begin the manufacture of the oligonucleotide. Because the synthesis cycle proceeds with a finite efficiency some material is lost while coupling each nucleotide monomer, as well as cleavage of the oligonucleotide from the solid support, and finally preparative purification following synthesis. The aggregate loss from each of these steps cause the final yield of the oligonucleotide to be less than the starting synthesis scale.