In general, the purity required for a specific application depends on the potential problems from the presence of failure sequences (n-1,n-2,....oligomers). For some applications, desalted oligos are satisfactory. For others, only HPLC or PAGE may yield oligos of sufficient purity. Yields will also vary according to the selected purification method.

Desalting:

Desalting removes residual by-products from the synthesis, cleavage and deprotecion procedure. Bionexus desalts every oligo.
Recommended applications: PCR, sequencing, probing, mobility shift, hybridization.

RP-HPLC:

For a higher level of purity all shorter non-extended synthesis can be removed. We recommend RP-HPLC for oligos shorter than 45 bases. The full-length product is 95-99% pure.
Recommended applications: Fingerprinting, mutagenesis, RT-PCR, in situ hybridization, microsatellite polymorphisms, primer extension.

PAGE

With the excellent resolution of PAGE for oligos longer than 45 bases. The full-length product is 95-99% pure. Recommended applications: gene synthesis, 32P-labelling.

Antisense / NMR grade:

If you want to use antisense oligos in cell culture, it is important to take care of the salts within the oligo sample. Our antisense oligos are precipitated and carefully dialyzed after a reversed phase purification.
Recommended applications: Phosphorothioates, large scales, NMR grade.

The scale of synthesis is the amount of 3' base that we start the synthesis with. It is NOT the expected final yield. The yield depends on the size of the oligonucleotide, the coupling efficiency(98-99%), and the base composition.

Theoretically the yield of the unpurified full length product is calculated using the formula 0.98ⁿ or 0.99ⁿ.

For PCR:
The unpurified 40 nmole scale yields approximately 800 to 4'000 reactions and the 0.2 umole scale yields 2'000 to 10'000 reactions.

For sequencing:
The unpurified 40nmol scale yields approximately 1'000 - 20'000 reactions depending on the sequencing technology.

Based on an unpurified 20-mer oligonucleotide:

Scale 0.04µmol 0.2µmol 1.0µmol 10µmol

OD 4 13 60 400

µg 132 429 1980 13200

nmol 20 65 300 2000

160 bases on the 0.2 umol scale. 60 bases on the 40 nmol scale. We do not increase our per-base charge to make longer oligonucleotides.

Bionexus uses industry standard beta-cyanoethyl phosphoramidite chemistry. This chemistry has been proven to be robust and to yield high quality oligos.

We monitor trityl yields and final synthesis yields. In addition, we monitor the overall synthesis quality of each oligonucleotide by gel electrophoresis and a digital gel image is supplied.

Bionexus /ABP custom oligonucleotides carry an unconditional guarantee and are backed by the highest quality technical support.

The label on every tube indicates the sequence, sequence number, nmol yield, oligo name, purification method and the modifications. We do not charge extra for the yield determination.

The oligonucleotides are shipped as lyophilized powder at room temperature in a clear 1.5 ml microcentrifuge tube. They have been deprotected in ammonia and desalted over Sephadex G-25. The Sephadex removes any excess ammonia, protection groups and very short oligonucleotide truncation products (5-mer or less).
The ammonia deprotection is necessary to remove the isobutyryl and benzoyl base protecting groups (these prevent unwanted side reactions during synthesis). Desalting is desirable to remove protecting groups and residual salts that can affect the biological activity of the oligonucleotide. Desalting is especially important for standard dideoxy sequencing and in vitro mutagenesis.

95% of our oligos are shipped out at the next day after receipt of your order (they have to pass the quality control) No shipment fee for A-priority.

• Sterile Water
• TE Buffer (10mM Tris-HCl, 1mM EDTA) pH 7.5-8.0

  Sterile, nuclease-free water is preferred.

  DNA Storage Conditions / Stability

• Lyophilized (-20 degrees C) / 6 months to several years

• Lyophilized (25 degrees C) / 2 months to 1 year

• Dissolved (-20 degrees C) / 1 month to 6 months

• Dissolved (25 degrees C) / 2 weeks to 3 months

  Microsynth recommends that for large experiments oligonucleotides be aliquotted into many tubes, lyophilized and stored in a -20 degree C freezer to maximize their shelf life. Oligonucleotides should not be frozen and re-thawed repeatedly because this process can lead to their physical degradation.

  Careful handling is recommended to avoid the possibility of bacterial contamination. Fluorescent oligonucleotides should be kept in the dark during storage, as light can slowly degrade the fluorescent moieties.
  
  
  

Definition of OD260

One OD unit of DNA is the amount of DNA that gives an absorbance reading of 1.0 at 260 nm for a sample dissolved in 1.0 ml total volume of ddH2O which is read in 1 cm quartz cuvette. (OD = "Optical Density"). For oligonucleotides, 1 OD is approximately 33 ug of DNA.

Melting Temperature (Tm) Estimation for Oligonucleotides.

The melting temperature (Tm value) of an oligonucleotide is dependent upon the length of the sequence, the G+C content of the sequence, and the type and concentrations of cation present, particularly sodium ion, Na+. A variety of formulas have been used for predicting Tm values. These tend to be fairly accurate for long DNA sequences, but are less accurate for oligonucleotides. The following formula is recommended for oligonucleotides ranging in length from 20 to 100 residues, and [Na+] concentrations ranging from 0.01 M to 1.0 M:

Tm (in degrees C) = 81.5 + 0.41(G%+C%) + 16.6 log[Na+] - 500/length

For example, for a 40-mer with a 55% G+C content in a 0.1 M NaCl solution:

Tm = 81.5 + 0.41(55) + 16.6 log[0.1] - 500/40

= 81.5 + 22.5 + 16.6(-1.0) - 12.5

= 74.9 degrees C

Annealing Temperature for PCR.

Calculate the melting temperature as above and subtract 5 to 10 degrees. If the two oligonucleotides have different melting temperatures, do NOT average the numbers. Use the lower number so that both of the oligonucleotides can anneal.

Molecular Weight

Molecular Weight = (A x 312.2)+(G x 328.2)+(C x 288.2)+(T x 303.2)-61 where A,C,G,T are # of A's, C's, G's and T's in an oligo.

Conversion Factor (Approximate Values)

OD units to ug Equivalence

1 OD260 unit of an oligo ~ 33 ug

1 OD260 unit of SS RNA = 40 ug

1 OD260 unit of dS DNA = 50 ug

OD Units to Nanomoles Equivalence

nmoles of oligo = 100 x OD/length of oligo

DNA Molar Conversions

1 ug of 1,000bp DNA = 1.62 pmol (3.24 pmoles of ends)

1 ug of pBR322 DNA = 0.36 pmol DNA

1 pmol of 1,000bp DNA = 0.62 ug

1 pmol of pBR322 DNA = 2.78 ug

Aliquotting.

We can economically supply a large number of aliquots of a sample, or sets of samples. This may help several labs share the same set of oligos.

Gene Synthesis.

We can produce entire genes of up to 15'000 base pairs in length to your custom specifications.

PCR Buffers:

To minimize your carry-over contamination in a polymerase chain reaction we deliver ready to use PCR-buffers (also prefilled in your PCR tubes).