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Type of purification and yield for oligonucleotide applications

Purification is recommended for all oligos greater than 40 bases in length and for many modified oligos. For demanding applications such as multiplex PCR, cloning, mutagenesis or antisense/RNAi methods, additional purification will significantly improve oligonucleotide performance. Every purified oligo up to 60 bases in length receives QC by capillary electrophoresis (CE) to document final purity.

SeGel Filtration

Size exclusion desalting process has been used on all Bio-Synthesis's oligos. For oligo >10 mers

rpCartridge and RP-LP Cartridge

This process is based on a specific Reverse-Phase purification by using the difference in hydrophobicity between the full-length product (with a 5' DMT group) and truncated sequences (without DMT groups). Bio-Synthesis recommends this purification for all applications where full-length or high reproducibility is essential, including: cloning, PCR, mutagenesis, probes.  The absence of salts and ammonium contamination, in addition to the absence of acid residues - which may occur after HPLC purification - make the cartridge purified oligonucleotides particularly suitable for use in cell culture. Two cartridge types:

  • The RP-cartridge for oligos from 8 to 39 bases long

  • The RP-LP cartridge for oligos from 40 to 80 bases long.

PAGE Purification

PAGE obtains extremely high purity, especially for unmodified oligos. This purification is based on molecular weight and charge, denaturing acrylamide gel electrophoresis provides a higher degree of purity than HPLC (93-95%).  PAGE purification is strongly recommended for oligos over 60 bases in length. For unmodified oligos, purity of >90% is routinely achieved.

HPLC Purification

  • HPLC is well suited to purify any modified oligo or unmodified sequences up to 60 bases in length and is the method of choice to purify larger amounts of oligos (i.e. > 1 µmol). Purity of > 85% is guaranteed for oligo up to 40 bases
  • Ion-exchange HPLC (IE-HPLC) is available to purify long oligos up to 60 bases. Anion-exchange resins separate oligos on the basis of charge. The resolution of this technique is very high but their use is limited to purification of rather short oligos. For unmodified oligos between 11-40, purity of > 90% is guaranteed.

For the most demanding applications for high purity oligonucleotide, Bio-synthesis offers

  • Dual HPLC Purification and Dual PAGE and HPLC Purification a combined purifications are applied for this service: HPLC + IEX HPLC purifications for 20 - 30mers oligos, and HPLC + PAGE purifications for 31 - 50mers oligos. Such combined purifications enable average of 98% purity by taking off most of base defective products or other impurities. Dual HPLC purification is used to purify Labeled Probes and Molecular Beacons with NHS Ester modifications.

RNase-Free HPLC Purification

Originally developed for isolating RNA oligos, RNase-Free HPLC Purification has been extended to DNA oligos for use when there is a high sensitivity to ribonucleases. RNase-Free HPLC purification is carefully performed in an RNase-free environment; reagents, equipment and lab surfaces are monitored for RNase contamination.

Cell Culture Purification

Oligo use in cell culture require multiple step of purification. Generally it consist of two HPLC purification steps and one sterile filtration step.

 

In vivo-Ready

Bio-Synthesis's In Vivo Ready siRNA option provides high quality oligo for gene knockdown that are ready for introduction into animals. Each siRNA strand is HPLC purified, dialyzed to ensure that the siRNA is salt free (conductance of <1 microsiemens for a 50 µM solution), sterile filtered, and endotoxin tested. Of course, each siRNA strand is also analyzed by MALDI-TOF mass spectrometry and analytical HPLC, and the efficiency of annealing is verified by PAGE.

Guide for selecting purification grade

  Gel
Filtration
Reverse-phase
Cartridge
Purification
HPLC
Purification
IEX HPLC
Purification
PAGE
Purification
Double
purifications
Gene amplification G G G G G E
Sequencing primers N G G G G E
Gene amplification for subcloning *1 N G/I G G G E
Degenerated DNA *2 N G I N N N
Mutagenesis N N G G E E
Gene synthesis N N G I E I
Oligo longer than 51mers N G E N E N
Oligo longer than 71mers N N G N E N
Modification (terminal) N N G I I N
Modification (insertion) N I G I I N
Modification (Fluorescent) N N G I N N
Phosphorothioate Oligo (S-oligo) N N G N N N
E Excellent
G Good
I Inquiry required (availability depends on sequence and modification)
N Not recommended
*1 HPLC or PAGE purification are recommended for oligos longer than 30mers or if require large amount of clones.
*2 Degenerated DNA means oligos with mixed base insertion.
* Please note that this table is only a guide without guarantee for your experiment. If anything goes wrong with using our product, please contact us.

Guaranteed yields for purified, unmodified DNA 20-50 bses in length**

  100 nmole 250 nmole 1 µmole 5 µmole 10 µmole
PAGE Purification 1 OD 2 ODs 10 ODs 50 ODs 100 ODs
HPLC Purification 1 OD 4 ODs 20 ODs 100 ODs 200 ODs
IE-HPLC Purification 1 OD 4 ODs 20 ODs 100 ODs 200 ODs
RNase-Free HPLC Purification 1 OD 4 ODs 20 ODs 100 ODs 200 ODs
Dual HPLC Purification 1 OD 2 ODs 10 ODs 50 ODs 100 ODs
Dual PAGE & HPLC 0.5 OD 1 OD 5 ODs 25 ODs 50 ODs

Guaranteed RNA Synthesis: Domestic Lead-time and Yield Guarantee*

  Desalted HPLC Purified
Scale 0.2µmol 1µmol 0.2µmol 1µmol
8-16 mer Yield Guarantee 5 OD 10 OD 2 OD 5 OD
17-30  mer Yield Guarantee 8 OD 15 OD 5 OD 10 OD
31-50  mer Yield Guarantee NA 15 OD 5 OD 10 OD
50-80  mer Yield Guarantee NA 30 OD 5 OD 10 OD
Domestic Lead Time 2-3 2-3 3-4 3-4

*Synthesis scale is not equal to guaranteed yield. Quantity varied depend on length and composition of the sequence, modifications and labeling.

Large RNA synthesis scale from mg to gram size are available, please call for quotation.