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A) Plasmid map of pGEM-T illustrates the position of T-overhangs used in T/A cloning and flanking T7 and <t>SP6</t> promoters. B) Percentage of cDNA insertion events in the sense orientation relative to the T7 promoter (yellow) and the SP6 promoter (blue) in constructs generated over the course of four CURE projects. The average across the projects is shown (checkered graph). C) Measured distribution of insert orientation from a cloning attempt of SMEST023171001.1 ( pumilio homolog) cDNA fragment assessed after the ligation and transformant selection steps. D) Bar graphs display orientation distribution of inserts after the ligation step from the cDNA fragment in (C; Gene 1) and two additional genes (SMEST004045004.1, Gene 2; and SMEST012773001.1, Gene 3), as well as their average (checkered bar). Orientation of Control Insert DNA ( luci ) included in the pGEM-T Vector System after the ligation step is also shown. Asterisks show statistical significance according to (* = p < 0.005) the chi-squared test and (*** = p < 0.0005) unpaired Student’s t- test.
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A) Plasmid map of pGEM-T illustrates the position of T-overhangs used in T/A cloning and flanking T7 and SP6 promoters. B) Percentage of cDNA insertion events in the sense orientation relative to the T7 promoter (yellow) and the SP6 promoter (blue) in constructs generated over the course of four CURE projects. The average across the projects is shown (checkered graph). C) Measured distribution of insert orientation from a cloning attempt of SMEST023171001.1 ( pumilio homolog) cDNA fragment assessed after the ligation and transformant selection steps. D) Bar graphs display orientation distribution of inserts after the ligation step from the cDNA fragment in (C; Gene 1) and two additional genes (SMEST004045004.1, Gene 2; and SMEST012773001.1, Gene 3), as well as their average (checkered bar). Orientation of Control Insert DNA ( luci ) included in the pGEM-T Vector System after the ligation step is also shown. Asterisks show statistical significance according to (* = p < 0.005) the chi-squared test and (*** = p < 0.0005) unpaired Student’s t- test.

Journal: bioRxiv

Article Title: Directionality bias in T/A cloning

doi: 10.64898/2026.02.11.705383

Figure Lengend Snippet: A) Plasmid map of pGEM-T illustrates the position of T-overhangs used in T/A cloning and flanking T7 and SP6 promoters. B) Percentage of cDNA insertion events in the sense orientation relative to the T7 promoter (yellow) and the SP6 promoter (blue) in constructs generated over the course of four CURE projects. The average across the projects is shown (checkered graph). C) Measured distribution of insert orientation from a cloning attempt of SMEST023171001.1 ( pumilio homolog) cDNA fragment assessed after the ligation and transformant selection steps. D) Bar graphs display orientation distribution of inserts after the ligation step from the cDNA fragment in (C; Gene 1) and two additional genes (SMEST004045004.1, Gene 2; and SMEST012773001.1, Gene 3), as well as their average (checkered bar). Orientation of Control Insert DNA ( luci ) included in the pGEM-T Vector System after the ligation step is also shown. Asterisks show statistical significance according to (* = p < 0.005) the chi-squared test and (*** = p < 0.0005) unpaired Student’s t- test.

Article Snippet: To tease this apart, a fragment corresponding to a planarian Pumilio homolog (SMEST023171001.1) was amplified from cDNA and ligated into pGEM-T. A portion of the ligation reaction was transformed into Escherichia coli JM109 cells, while another portion of the ligation reaction was used directly as template to sequence across the insertion site between the T7 and SP6 promoter using third generation long read sequencing (PCR-EZ, Azenta).

Techniques: Plasmid Preparation, Cloning, Construct, Generated, Ligation, Selection, Control