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Oligonucleotide Synthesis

Gene Synthesis & Molecular Biology

Sanger Sequencing

Next Generation Sequencing

Genotyping & Gene Expression

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Combinatorial Libraries

 

 

Gene libraries are a pool of DNA sequences, based on a single input sequence, harbouring mutations at defined positions.

 

 

 

 

 

 

 

Applications

 

Combinatorial Gene Libraries are very useful for high-throughput screening and allow to

  • Optimise the binding characters of antibodies
  • Optimise protein properties (e.g. protein stability / activity)
  • Optimise for improved expression of your protein
  • Find the best DNA sequence for improved protein binding
  • Improve ribozyme activity

 

Choose between different kinds of Combinatorial Libraries:

  • Linear library
  • Cloned library
  • In addition to Combinatorial Libraries Eurofins also other library tyes

 

Further details about the different library types can be found below

Complexity of a library

 

Cloned libraries from Eurofins are delivered with a maximum diversity of 108 variants. If too many wobble positions are selected it is even not statistically possible to deliver all clones. Here are some examples:

 

...GCT NNN AGC T...

  • 64 possible variants (4x4x4 = 43)
  • All amino acids at one position

 

...GCT NNK AGC T...

  • 32 possible variants (21x42)
  • NNK = all amino acids excluding 2 stop codons
  • Reduced complexity

 

...GCT NNN NNN NNN NNN NNN AGC T...

  • 15 N = 415 = 109 possible variants
  • All amino acids at 5 positions - variantion is very high (109). Consider reducing the complexity of the library.
  • Using 5xNNK would reduce the complexity to 3*107

 

 

 

 

 

 

 

Product Overview & Selected Publications

 

Linear libraries

Basic GeneStrands library - starting from €309*

 

Linear Libraries are PCR products harbouring a certain number of degenerated positions. During oligo synthesis degenerated positions are introduced.

 

Specifications

  • Standard sequence up to 1000 bp
    moderate GC content (35-75%), without extensive repeats (>25bp) or critical hairpin structures and without extensive homopolymer stretches (>18bp)
  • Scale up to 500 ng
  • Delivery format: lyophilised PCR product

 

Quality controls

  • Single strand sequencing of the linear library

 

Incorporate wobble bases into GeneStrands - get a linear DNA library at very competitive prices

 

At Eurofins Genomics you pay per stretch of degenerated positions, not for every single position. This makes our linear libraries very attractive. Always keep in mind that complexity can increase quickly into astronomically high numbers. Therefore it is a good idea to keep complexity of your library as low as possible.

 

We also have mention one point: we cannot entirely exclude the possibility that, after cloning, you might find clones that harbour additional mutations elsewhere in the sequence. This is due to the nature of the gene synthesis process, where we use highest quality oligos; but even the best oligos do harbour mutations at very low frequencies (about 1 in 2000 bp). However, this might even result in serendipity, maybe you find the unexpected variant, that otherwise would not have been identified. Serendipity means "making discoveries by accident" or "pleasant surprise".

 

If you absolutely need 100% correct sequence outside the degenerated stretches, just contact us!. We will then discuss the project with you and offer a solution for your needs.

 

 

Cloned libaries

Ready-to-Use

 

A cloned library basically is a linear library that has been cloned into a plasmid.

 

We offer cloning into our Gene Synthesis standard vector pEX-A2 (map) or in a vector of your choice. We routinely deliver plasmid libraries at complexities of up to 108.

 

Specifications

  • Standard sequence up to 1000 bp
    moderate GC content (35-75%), without extensive repeats (>25bp) or critical hairpin structures and without extensive homopolymer stretches (>18bp)
  • Diversity up to 108 variants per library
  • Scale approx. 500 µg
  • Delivery format: complete library in one tube

 

Quality controls

  • Double strand sequencing of the cloned library
  • Single strand sequencing of 10 single clones

 

QC criteria

100% sequence homology of the cloned library to the expected sequence, mixed peaks at degenerate positions. Please note that for some libraries due to the degenerate positions a higher-than-usual background elsewhere in the sequence can occur.

 

Additional options

  • Sequencing of 96 individual plasmids
  • Statistical data analysis (Chi2 value) of 96 clones, see graph on the top right
  • Glycerol culture of the cloned library

 

Important information

We cannot entirely exclude the possibility that, after cloning, you might find clones that harbour additional mutations elsewhere in the sequence. This is due to the nature of the gene synthesis process, where we use highest quality oligos; but even the best oligos do harbour mutations at very low frequencies (about 1 in 2000 bp). However, this might even result in serendipity, maybe you find the unexpected variant, that otherwise would not have been identified. Serendipity means "making discoveries by accident" or "pleasant surprise".

 

If you absolutely need 100% correct sequence outside the degenerated stretches, just contact us!. We will then discuss the project with you and offer a solution for your needs.

 

Tip

If you add a promoter and a terminator to your library sequence, you can get an expression plasmid ready for high-throughput screening.

 

Other libaries

Libraries, Libraries, Libraries

 

In addition to the Combinatorial Libraries we also offer:

 

  • (Ala) Scanning Libraries
    Identify essential amino acids (aa) positions within an ORF (coding sequence
  • Site Saturation (Mutagenesis) Libraries
    Test all possible amino acids at one site (triplet / codon)
  • Truncation Libraries
    Shortened versions of a full-length gene

 

If you are interested in one of these libraries, just contact us!

 

 

 

Literature

 

 

 

 

 

 

 

 

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Quality is important for us

Our products and services are produced and performed under strict quality management and quality assurance systems.

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