Degenerated Libraries
(up to 1011 variants)
degeneration of multiple codons
The Problem
Directed evolution strategies are perhaps the most efficient methods of creating proteins with improved or novel properties. Until recently, conventional fragmentation-based DNA shuffling protocols or error-prone PCR were the methods of choice to create suitable libraries for in vitro evolution assays. However, these strategies either depend on the availability of highly homologous genes or create huge random libraries with a vast amount of non-functional variants.
The Solution
De novo library synthesis allows for a true rational design that is based on available information about the protein and offers a maximum of flexibility. While leaving most of the coding framework untouched and accurate, it is possible to degenerate only those protein positions that are likely or known to contribute significantly to the protein's function.
Deliverables
Degenerated libraries can be delivered in different formats:
- non-amplified library: Up to 10<sup>11</sup> molecules as verified by real-time PCR.
- amplified library: 5 µg of linear DNA ready to clone via 5' and 3' restriction sites.
- cloned library: 10 µg of plasmid DNA. Library subcloned into customer's vector. Includes glycerol stock of total library transformants.
Quality control
Amplified and cloned library pools will be bulk sequenced. Additionally, a peer group of 48, 96 or 192 individual transformants will be sequence verified. The statistical analysis of the obtained data includes:
- determination of correctness of the non-degenerated part of the sequence
- even distribution of nucleotides at the degenerated sites
- library size via real-time PCR to verify the number of distinct molecules in the non-amplified library
GENEART offers a guaranteed threshold for all of these three critical parameters.
Fill out our online quotation form to get a quote for your specific project.
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