 | Acta Crystallographica Section D Acta Crystallographica Section D BIOLOGICAL CRYSTALLOGRAPHY |
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![[Figure 2]](gx5081fig2mag.jpg)
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Figure 2
Generic workflow for HTP cloning and expression screening. The scheme is based on the current procedures of the Oxford Protein Production Facility and shows that both cloning and expression screening can be carried out in two working weeks with certain steps automated. PCR reactions for both amplifying the target-gene sequences and for screening mini-preps following cloning (robotic screen of plasmid DNA mini-preps) are carried out in a semi-automated procedure using a PCR cycler integrated into a liquid- and microplate-handling system (MWG Theonyx). Small-scale plasmid DNA mini-preps are prepared automatically using the Qiagen 8000 instrument and associated protocol/reagents. Expression vectors are verified by PCR screening and sequencing on one strand only from a single primer (T7 forward). Transformation of E. coli strains both for cloning and expression screening is carried out manually with cells plated out on standard 24-well plates (15 mm diameter well) to give a density of approximately 10–20 colonies per well determined empirically. Colony picking and culture in deep-well blocks is also carried out by hand. Expression screening is semi-automated with small-scale Ni–NTA purification of soluble proteins carried out using the Qiagen 8000 instrument and associated protocol/reagents. |
![[Figure 2]](gx5081fig2.jpg)
| BIOLOGICAL CRYSTALLOGRAPHY |
ISSN: 1399-0047
