High throughput cloning with restriction enzymes.
The systematic structural analysis of many target proteins involves generating expression clones in high throughput. This requires robust laboratory procedures and benefits from laboratory automation and data management systems. This chapter gives an overview of the Protein Structure Factory, a structural genomics project focusing on human proteins, and presents the authors' method for cloning bacterial expression clones with the restriction enzymes BamHI and NotI and compatible enzymes. PCR amplification, product purification and digestion and vector ligation were adapted to the 96-well microtiter plate format.
Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Protein Structure Factory, Berlin, Germany.
Volker
V
Sievert
Asgar
A
Ergin
Konrad
K
Büssow
D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases