Page 36 - Macromolecular Crystallography

HIGH-THROUGHPUT CLONING, EXPRESSION, AND PURIFICATION 25

solubility of the cloned DNA product if they form homology between extensions on the PCR product
part of the translated sequence. This can be avoided and the ends of a linearized vector; the optimal
by positioning the att sites outside of the ORF, but length for these homologous sequences is around
some of the flexibility of the system is lost since only 15 base pairs. Once these homologous extensions
a single fusion format is possible. have been incorporated into the PCR product no
further processing of the insert is required prior
to the In-Fusion™ reaction, in contrast to PCR-LIC
2.2.1.3 In-Fusion™ methods (Protocol 2.2). The main advantage of the
The In-Fusion™ method is both insert sequence- In-Fusion™ method is that the user can define the
independent and enables cloning of PCR prod- exact sequence of these primer extensions without
ucts directly into any cloning or expression vector the limitations on base and codon usage inherent in
(Fig. 2.1c). The mechanism of the reaction has not the T4 polymerase-based LIC system. With minor
been fully reported but relies on the presence of vector modifications (e.g. insertion in the cloning

(a) LIC-PCR (custom) (b) Gateway TM (two-step)

Linearize, T4 T4 polymerase treat Purify attB-‘tagged’
polymerase treat in presence of PCR product (or
in presence of ‘lock’ complement of pDONOR221 (attP) DEST expression
dNTP (~50 min) and vector ‘lock’ dNTP clone)
purify (~50 min) and purify.

BP Clonase reaction 1 Hour at
25˚C, Proteinase K ‘kill’ reaction at
Anneal for 5 min 37˚C for 10 min
at room temperature
Entry clone (attL)
ccdB
+

Transform into E.coli with
8
Transform into E.coli competency >10 c.f.u./µg DNA,
8
(competency >10 c.f.u./µg DNA). Normally plated on 4 X 24-well LB Agar
Plasmid ‘backbone’ repaired/ligated Plates supplemented with Kanamycin
by E.coli, Normally in 24-well plates

Pick colonies, grow, prepare
Pick colonies, grow, prepare plasmid, PCR screen for insert
plasmid. PCR screen for insert

Cloning-Ready/
Entry Clone
Expression-ready plasmid minipreps
plasmid mini-preps

Figure 2.1 Continued