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Purification

Purification
 
 
Oligonucleotides are assembled via solid phase synthesis. Thereby nucleotides are attached according to the sequence of choice leading to chain-growth. It is generally known that chemical reactions never get at 100% conversion. In state-of-the art oligonucleotide synthesis coupling yields of up to 99.5% are achieved. With the unreacted 0.5% of the strands, chain assembly stops and truncated sequences form as side products.
Depending on the intended application it is advantageous to remove these shorter sequences from the full-length product. Therefore, Microsynth offers various types of purification methods.

Overview

Desalted

All our oligos are at least desalted to largely remove residual low molecular by-products arising and accumulating from the frequent chemical reactions during synthesis. Such purification is sufficient for oligonucleotides shorter than 30 nt and/or oligonucleotides used for non-critical applications such as PCR, sequencing, probing, mobility shift or hybridization. However, desalted oligos are not recommended for use in molecular cloning projects.

Potential Applications:

  • PCR
  • DNA sequencing
  • Probing
  • Mobility shift or hybridization

RP-HPLC Purification

Oligos <50 nt in length can be well purified via RP-HPLC (reverse-phase high-performance liquid chromatography). Through this purification approach, preferably residual, n-x truncated oligos (lacking the hydrophobic DMT protection group at the 5’ end) are removed. This results in a ≥85% purity of the targeted oligonucleotide. RP-HPLC is useful for a higher level of purity required for more demanding applications such as cloning, DNA fingerprinting, real-time PCR, FISH, etc.

Potential Applications:

  • Molecular cloning
  • DNA fingerprinting
  • Real-Time PCR and digital PCR*
  • FISH

* Microsynth primer and qPCR probes are compatible with all commercially available supermixes

IEX-HPLC Purification

IEX-HPLC (ion-exchange high-performance liquid chromatography) is a preferred purification method for longer oligonucleotides (40-80 nt). Through this purification approach, residual n-x truncated oligos are removed efficiently. Whereas RP-purification yields very good results for oligos < 50 nt, IEX purification is superior for longer oligonucleotides. (40-80 nt). For oligos with this length IEX results in ≥85% purity of the targeted oligonucleotide. IEX is useful for a higher level of purity of long oligonucleotides required for more demanding applications such as direct cloning or NGS applications.

Potential Applications:

  • Molecular cloning (direct cloning)
  • NGS Applications

HPLC Purification & Dialysis

Dialysis as an add-on to HPLC is recommended if oligos need to be present in a physiological state. When performing in vivo experiments (e.g. in mice) this purity level is strongly recommended.

Potential Applications:

  • Antisense experiments
  • Cell culture studies
  • Physical Chemistry and Structure Analysis (NMR, MS etc.)

PAGE Purification

Polyacrylamide gel electrophoresis (PAGE) purification is generally necessary for long oligos (>50 bases) and for all those primers with critical 5' sequences (restriction endonuclease sites, RNA promoters). It is the best method to differentiate full-length oligos from aborted sequences (n-1 oligos), based on size, conformation and charge. PAGE purification has an excellent resolution and yields a product that is, on average, ≥95% pure. In this context, it is important to note that the purity level declines with increasing length of the oligonucleotide, and this is particularly true for oligos up to 120 bases. PAGE purification is highly recommended for sensitive experiments such as cloning, mutagenesis, DNA fingerprinting, in situ hybridization, gene synthesis, etc.

Potential Applications:

  • Molecular cloning
  • Mutagenesis
  • DNA fingerprinting
  • In situ hybridization
  • Gene synthesis

DNA Yields

 
Desalted DNA Oligos
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics 13 - 60 2 10 1
0.04 µmol 13 - 80 3 15 1
0.2 µmol 6 - 1504 10 50 1
1.0 µmol 6 - 80 50 250 1
15 µmol 13 -60 700 3'500 2
 
RP-HPLC Purified DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol 13 - 50 1 5 2
0.2 µmol 6 - 50 3 15 2
1.0 µmol 6 - 50 15 75 2
15 µmol 6 - 50 300 1'500 3
 
IEX-HPLC Purified DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]5
Genomics not available
0.04 µmol not available
0.2 µmol 40 - 80 2 3.3 3-5
1.0 µmol 6 10 3-5
15 µmol not available
 
HPLC Purified & Dialysed DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol 8 - 50 3 15 3
1.0 µmol 8 - 50 15 75 3
15 µmol 8 - 50 200 1'000 4
 
PAGE Purified DNA Oligos
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol 13 - 80 0.5 2.5 2
0.2 µmol

8 - 80

81 - 1504

2

0.5

10

2.5

 2
1.0 µmol 8 - 80 7 35 2
15 µmol not available
 
 
1 The synthesis scale represents the initial amount of 3' bases (starting material).
2 Our guaranteed and average yields are measured in OD and are valid only for unmodified oligos >20mer.
3 Yields indicated in nmol represent an example calculation for a 20mer. For this calculation the following rule of thumb equation was applied: nmol of oligo = OD x 100/length of oligo. Please note that this calculation is based on sequences with virtually homogenous distribution of the 4 DNA bases; it may vary for sequences with high GC contents >70% etc.
4 Oligos longer than 150 DNA bases on request (we would like to discuss the proposed experiment/application with you beforehand in order to guarantee the best possible outcome)
5 Yields indicated in nmol represent an example calculation for a 60 nt DNA Oligo. Formula: nmol= OD*100/length of the Oligo

RNA Yields


Desalted RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol 10 - 30 4 21 2
0.2 µmol 10 - 50 8 35 2
1.0 µmol 10 - 50 18 80 2
15 µmol 10 - 40 400 1'800 3
 
HPLC Purified RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol 10 - 30 1 5 2
0.2 µmol 10 - 50 3 15 2
1.0 µmol 13 65 2
15 µmol 10 - 40 300 1'500 4
 
HPLC Purified & Dialysed RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol 10 - 50 2 10 4
1.0 µmol 9 45 4
15 µmol 10 - 40 200 1'000 4
 
PAGE Purified RNA Oligos
Synthesis scale1 Length Restriction Guaranteed Yield2 Production Time [wd]
[OD260] [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol 10 - 50 1 5 2
1.0 µmol 10 - 50 6 30 2
15 µmol not available

 

1 The synthesis scale represents the initial amount of 3' bases (starting material).

2 Our guaranteed and average yields are measured in OD and are valid only for unmodified oligos >20 and <40 nucleotides.
3 Yields indicated in nmol represent an example calculation for a 20mer. For this calculation the following rule of thumb equation was applied: nmol of oligo = OD x 100/length of oligo. Please note that this calculation is based on sequences with virtually homogenous distribution of the 4 RNA bases.