Fig 1.
Scheme of the QuickLib method for cloning degenerate oligonucleotides into plasmids.
A plasmid is initially PCR-amplified using an asymmetric pair of primers sharing complementary 5’ ends (blue). The randomized sequence in the long primer is coloured in red. The library of linearized synthetic plasmids is then circularized by the combined actions of 3 enzymes and the methylated starting matrix is selectively removed by digestion with DpnI.
Fig 2.
Circularization of linear plasmid library and removal of starting matrix.
(a) Time course of the circularization reaction: a mix of four enzymes (T5 exonuclease, DNA polymerase, DNA ligase and DpnI) was added to the amplified linear plasmids and incubated for one hour at 50 C. The amount of T5 exonuclease was reduced 4-fold compared to Gibson’s protocol. Circularization products (left side) are also analyzed by restriction with AvaI (right side). The band at 3.6 kb (black arrow) is only present when the plasmids are sealed. (b) DpnI is cleaving the starting matrix at 50 C while the synthetic PCR product is resistant to its action.
Fig 3.
Dependence of 5’ homology length of primer pairs on the efficiency of QuickLib assembly.
(a) A long degenerate primer (84 nt) and different short primers (27 nt) sharing the same overall length but with variable homology length were tested as primer pairs (full sequences are given in S1 Table). The melting temperatures of duplexes between forward and reverse primers, i.e. between the homology regions that must be annealed for the circularization step, are listed. (b) Full plasmid PCRs gave similar yields of linear plasmids. (c) After circularization/purge reaction, the amount of circularized product (red arrow) was estimated by AvaI restriction. (d) Overall efficiency was evaluated by measuring the number of colony forming units (cfu).