Download Expresso Solubility and Expression Screening System

Expresso® Solubility and Expression Screening System
Detailed Protocol
1. Preparation of Insert DNA
1.1. Primer Design
To clone with Expressioneering™ Technology, the target DNA must first be amplified with
primers that add flanking sequence identical to those found at the ends of linear pSol vectors
(Figure 3).
Forward Primer Features
 The forward primer should be of the general structure:
5’-AAT CTG TAC TTC CAG GGT XXX XXX XXX XXX XXX XXX …-3’

The required sequence at the 5’ end of the forward primer includes 6 of the TEV
recognition site codons. This sequence is also found at one end of all of the pSol
vectors.

The 3’ end of the forward primer anneals to the bottom strand of the target gene,
usually beginning with the second codon. The length of complementarity to the target
gene will depend on the sequence.

Because each Expresso pSol vector contains an ATG initiation codon immediately
preceding the 6xHis codons, the ATG initiation codon from the target gene is not
needed.
Reverse Primer Features
 The reverse primer should be of the general structure:
5’-GTG GCG GCC GCT CTA TTA XXX XXX XXX XXX XXX XXX …-3’

The required sequence at the 5’ end of the reverse primer matches 18 bases of the
downstream end of the pSol vectors.

The 3’ end of the reverse primer anneals to the top strand of the target gene, usually
including the last codons prior to the stop codon of the coding region. The length of
complementarity to the target gene will depend on the sequence.

Because the pSol vectors include in-frame stop codons, it is not necessary to include
the stop codon of the target gene.
Factors affecting the length of the target-specific portion of the primers include: GC content,
Tm, and potential for formation of hairpins or primer-dimers. We recommend that targetspecific portion of each primer be designed with a Tm of ~60°C. The annealing temperature used
in amplification may be adjusted to accommodate primers with higher or lower Tm.
Figure 3. Primers for cloning into the Expresso pSol Vectors.
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MA158 Rev B