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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 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 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

 

HPLC Purification

Oligos <50 bases in length can be well purified via Reverse Phase HPLC. 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 90-95% 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
  • FISH

 

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, 95-99% 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 >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 Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics 13 - 50 2 10 8 40 1
0.04 µmol 13 - 80 3 15 9 45 1
0.2 µmol 6 - 1504 10 50 20 100 1
1.0 µmol 6 - 80 50 250 80 400 1
15 µmol 13 - 60 700 3‘500 1000 5000 2
 
 
 
HPLC Purified DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics not available
0.04 µmol <51 1 5 4 20 2
0.2 µmol 3 15 7 35 2
1.0 µmol 15 75 26 130 2
15 µmol 300 1'500 500 2500 4
 
HPLC Purified & Dialysed DNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol

<51

3 15 6 30 3
1.0 µmol 15 75 20 100 3
15 µmol 200 1'000 400 2000 4
 
PAGE Purified DNA Oligos
Synthesis scale1 Length Restriction Guaranteed Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics not available
0.04 µmol 13-80 0.5 2.5 6 30 2
0.2 µmol 8-80
81-1504
2
0.5
10
2.5
8
4
40
20
2
1.0 µmol 8-80 7 35 20 100 2
15 µmol not available

 

1 The synthesis scale represents the initial amount of 3' bases (starting material).
2 Guaranteed and average yields in OD are valid for unmodified oligos >20mer only.
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)

RNA Yields

 
Desalted RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics not available
0.04 µmol 10 - 30 4 21 6 28 2
0.2 µmol 10 - 504 8 35 10 45 2
1.0 µmol 10 - 504 18 80 22 100 2
15 µmol 10 - 40 400 1'800 800 2'200 3
 
HPLC Purified RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics not available
0.04 µmol 10 - 30 1 5 2 10 2
0.2 µmol 10 - 80 3 15 5 25 2
1.0 µmol 13 65 17 85 2
15 µmol 10 - 40 300 1'500 360 1'800 4
 
HPLC Purified & Dialysed RNA Oligos 
Synthesis scale1 Length Restriction Guaranteed Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol 10 - 80 2 10 3 15 4
1.0 µmol 9 45 11 55 4
15 µmol 10 - 40 200 1'000 250 1'250 4
 
PAGE Purified RNA Oligos
Synthesis scale1 Length Restriction Guaranteed Yield Average Yield Production Time [wd]
[OD260]2 [nmol]3 [OD260]2 [nmol]3
Genomics not available
0.04 µmol not available
0.2 µmol 10 - 504 1 5 1 5 2
1.0 µmol 10 - 504 6 30 7 30 2
15 µmol not available

 

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

2 Guaranteed and average yields in OD are valid for unmodified oligos >20 and <40 nucleotides only.
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.

4 Oligos longer than 50 RNA nucleotides can be produced in combination with HPLC purification.