General Considerations For Oligonucleotide Design
80-100 bp oligonucleotides can be generated as single molecules, or pairs of oligonucleotides that are completely overlapping or partially overlapping. The type of oligonucleotide recommended depends on the type of mutation and the distance from the CORE cassette integration site a mutation is desired.
Longer oligonucleotides lead to increased transformation efficiency. Fully complementary oligonucleotides pairs lead to 5-10 fold increase in efficiency compared to single oligonucleotides and are recommended for all applications. However, they provide a small window of mutagenesis of only 20-40 bases from the CORE cassette. To increase the window of mutagenesis, oligonucleotide pairs with a 20 bp overlap can be used, and these allow up to 100 bp upstream and downstream of the CORE integration site to be targeted. However, they transform approximately 6 times less efficiently. To increase the efficiency, partly overlapping oligonucleotides can be extended in vitro.
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