Fig. 3

Functional pUC19 vector dephosphorylation assays. a. EcoRI-linearized pUC19 DNA, subjected to dephosphorylation and self-ligation. Lane M, GeneRuler 1 kb DNA Ladder; lane K1, undigested pUC19 DNA; lane K2, EcoRI-linearized pUC19 DNA; lane LC (ligation control), non-dephosphorylated, selfligated EcoRI-linearized pUC19 DNA; lane 1, EcoRI-linearized pUC19 DNA dephosphorylated with 0.01 colorimetric U of His6-BAP and selfligated; lane 2, with 0.005 U; lane 3, with 0.0025 U; lane 4, with 00125 U. b EcoRI-linearized pUC19 DNA, subjected to dephosphorylation and self-ligation—fine-tuned His6-BAP concentrations. Lane M, GeneRuler 1 kb DNA Ladder; lane K1, undigested pUC19 DNA; lane K2, EcoRI-linearized pUC19 DNA; lane LC, non-dephosphorylated, selfligated EcoRI-linearized pUC19 DNA; lane 1, EcoRI-linearized pUC19 DNA dephosphorylated with 0.006 colorimetric U of His6-BAP and selfligated; lane 2, with 0.005 U; lane 3, with 0.004 U; lane 4, with 0.003 U; lane 5, with 0.002 U; lane 6, with 0.001 U. c KpnI- or SmaI-linearized pUC19 DNA, subjected to dephosphorylation and self-ligation. Lane M, GeneRuler 1 kb DNA Ladder; lanes K1, undigested pUC19 DNA; lane K3, KpnI-linearized pUC19 DNA; lane LC, non-dephosphorylated, selfligated KpnI-linearized pUC19 DNA; lane 1, KpnI-linearized pUC19 DNA subjected to dephosphorylation with 0.05 colorimetric U of His6-BAP and selfligation; lane 2, as in lane 1, except that 0.01 U His6-BAP were used; lane 3, 0.005 U; lane K4, SmaI-linearized pUC19 DNA; lane LC, non-dephosphorylated, selfligated SmaI-linearized pUC19 DNA; lane 4, SmaI-linearized pUC19 DNA dephosphorylated with 0.05 colorimetric U of His6-BAP and selfligated; lane 5, with 0.01 U; lane 6, with 0.005 U