 | Acta Crystallographica Section D Acta Crystallographica Section D BIOLOGICAL CRYSTALLOGRAPHY |
journal menu
![[Figure 1]](bw5097fig1mag.jpg)
|
|
Figure 1
Nested PCR and recombinant cloning using the Gateway technology. Nested PCR is performed so that the resulting constructs comprise the target ORF flanked by the attB recombination sequences. Gene-specific primers used in the first round of PCR contain a 12-base-pair overlap with the generic primers used in the second round. The generic forward primers include 21 nucleotides that encode the rTEV peptide-recognition site followed by three spacer amino acids (SGA) for optimal rTEV cleavage of the recombinant protein. The PCR product is subcloned into pDONR221 (BP reaction) by incubating with BP Clonase for ≥1 h at 298 K. Following selection of a positive Entry clone (detailed in Fig. 2 ![[link]](../../../../../../logos/arrows/d_arr.gif) ), the ORF is transferred into at least two destination vectors (LR reaction). Entry clones are incubated with pDEST17 (or pDEST15) and LR Clonase for ≥1 h at 298 K. |
![[Figure 1]](bw5097fig1.jpg)
| BIOLOGICAL CRYSTALLOGRAPHY |
ISSN: 1399-0047
