Skip to main content
Fig. 1 | BMC Biotechnology

Fig. 1

From: Reliable handling of highly A/T-rich genomic DNA for efficient generation of knockin strains of Dictyostelium discoideum

Fig. 1

Knockin vector construction by a linear DNA cloning system. a 3-step construction of the 3′-tagging vector. Step 1: Preparation of pJAZZ vector harbouring A/T-rich 3′ recombination. Step 2: Assembly of the knockin vector by 4-piece ligation. Step 3: Release of knockin cassette by NotI digestion. b Design of GFP knockin vector for DDB_G0273397/carA-1 harbouring 1.0 kb each of 5′ and 3′ recombination arms. c, d Stable cloning of A/T-rich 3′ UTR/terminator of carA-1 by linear cloning system. 1 kb of 3′ UTR/terminator of carA-1 were blunt cloned into pBluescript (c) and pJAZZ vector (d). Release of the insert in randomly selected 6 DNA clones was checked by restriction enzyme digestion with XhoI and SpeI for pBluescript and with NotI for pJAZZ (these are the multiple cloning sites on each vector). Variable size of released fragments in C indicates deletions of circular plasmids. Appropriate size of inserts (arrow in d) were released from all the 6 clones of pJAZZ vector. The lane for negative control (Vector) was loaded with NotI-digested pJAZZ vector carrying no insert. Arrow heads represent the long and short arm of NotI-digested pJAZZ vector. e Four DNA fragments as depicted in B were subjected to directional ligation. f DNAs from randomly selected TSA E. coli clones were digested with NotI to excise the 4.5 kb of assembled knockin cassette (arrow). g Appropriate DNA assembly in 4 clones (same as in f) was detected by PCR for fragment ligation between 2 and 3 (upper column, 2 + 3) or fragment 3 and 4 (bottom column, 3 + 4). Primer position was depicted in (b). All the molecular marker was1 kb DNA ladder

Back to article page