Journal: PLoS ONE
Article Title: A Novel Selection Marker for Efficient DNA Cloning and Recombineering in E. coli
doi: 10.1371/journal.pone.0057075
Figure Lengend Snippet: ( A ) Larger colonies were formed by cells transformed with larger mfabI plasmids on plates. Shown are colonies of DH5α cells transformed by pF2 ( i & iv ), pF2-DTA-Rosa26 ( ii & v ), or pF2-DTA-Rosa26-Insert ( iii & vi ) and grown at 37°C ( i, ii & iii ) or 32°C ( iv, v & vi ) on 1 µM triclosan LB plates; insert in each panel is the low-magnification view of the whole plate. ( B ) Higher cell densities obtained in overnight cultures of larger mfabI plasmid transformants. Two colonies per group were picked from 32°C-grown plates and grew in 1 µM triclosan LB broth at 37°C for 17 hrs before O.D. measurements. *: p<0.05 **: p<0.01 in one-tailed, unpaired t-test (N = 2). Error bars show standard deviations. ( C ) Plasmid DNA yields from overnight cultures. Plasmid DNA was extracted from cultures used in (B). Error bars show standard deviations (N = 2). ( D ) Restriction enzyme (XhoI/XbaI)-digested plasmid DNA used in (C). Molar equalized amounts of DNA were digested. All clones showed expected correct digestion patterns (refer to maps in ). M is DNA molecular weight marker lane (Invitrogen 1 kb plus DNA ladder); numbers denote kb bands.
Article Snippet: Correct clones were identified by restriction enzyme digestion of miniprep DNA and confirmed by sequencing with Fab-BstE2-F and Fab-BssH2-R. pF-DTA was generated by recombineering with fragments from pDTA, which has the EF1a-DTA negative selection marker cloned into pBS backbone, cut by Apa LI deleting ampicillin resistance marker and pF by Afl III/ Dra III containing fabI . pF2-DTA was generated by cloning mfabI into pF-DTA by Fsp I/ Apa LI. pF2-DTA-Rosa26 was generated by inserting fragment from pRosa26-1 (Addgene plasmid 21714) cut by Eco 53kI/ Sal I into the Nru I/ Sal I sites in pF2-DTA.
Techniques: Transformation Assay, Plasmid Preparation, One-tailed Test, Clone Assay, Molecular Weight, Marker
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