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Purification
 
Oligonucleotides are assembled via solid-phase synthesis. In that way, nucleotides are attached according to the sequence of choice leading to chain growth. It is generally accepted that chemical reactions never reach 100% conversion. However, 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

PAGE Purification

Polyacrylamide gel electrophoresis (PAGE) purification is generally speaking 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 for differentiating 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 the 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

 
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

 
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.