Journal: PLoS ONE
Article Title: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes
Figure Lengend Snippet: Assembly of a GFP construct from 10 plasmids. (A) Construct maps. Input modules contain a core region C flanked by BsaI restriction sites in opposite orientation composed of a recognition site (B, ggtctcn, B , ngagacc) and a 4 nucleotide cleavage site (boxes flanking the core region). pX-LacZ, acceptor vector. pGFPi, resulting construct. Restriction sites for AvrII and XmaI are shown as white arrows. (B) Ethidium bromide-stained gel with products obtained by restriction-ligation of the 9 input module plasmids. M: GeneRuler 1kb DNA Ladder Plus from Fermentas. Restriction-ligation was performed at 37°C for 3 (lane 3h) or 6 hours (lane 6h) or with 25 cycles (2 min 37°C+5 min 16°C, lane 25) or 50 cycles (lane 50), and without BsaI enzyme (lane nb). The arrow indicates the 1.17 kb linear assembled GFP gene product. (C) Ethidium bromide-stained gels of 72 minipreps digested with XmaI and AvrII (expected fragment sizes: 4.6 kb, 945 and 555 bp), obtained from restriction-ligations performed for 6 h 37°C (6 h), for 25 or 50 cycles (25×/50×), with normal ligase (nl) or high concentration ligase (hcl). Numbers indicate minipreps with an incorrect restriction pattern, and stars indicate constructs that consist of dimers (same restriction pattern as monomers). V, vector pX-lacZ.
Article Snippet: A restriction-ligation was set up by adding into a single tube 50 ng of each of the 27 trypsinogen fragment constructs , 50 ng of vector, 10 units of BsaI enzyme (NEB) and 3 units of T4 DNA ligase (Promega) in a total volume of 15 microliters in ligation buffer (Promega).
Techniques: Construct, Plasmid Preparation, Staining, Ligation, Concentration Assay