Journal: BMC Genomics
Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity
Figure Lengend Snippet: Controlling of partial cleavage of genomic DNAs by TthHB27I under cofactor/analogue-induced ‘star’ activity conditions. E. coli and λ genomic DNAs were digested with TthHB27I under cofactor/analogue-induced ‘star’ conditions, while varying: temperature, incubation time and TthHB27I amount. 0.5 μg of DNAs were digested with TthHB27I in REase buffer supplemented with 100 μM SIN and 25% (v/v) DMSO. a Cleavage control by reaction temperature. Reactions conducted with E. coli DNA digested with 2 U of TthHB27I for 1 h at temperatures from 30 °C to 80 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction at 30 °C; lane 2, 35 °C; lane 3, 40 °C; lane 4, 45 °C; lane 5, 50 °C; lane 6, 55 °C; lane 7, 60 °C; lane 8, 65 °C; lane 9, 70 °C; lane 10, 75 °C; lane 11, 80 °C. b The same as ( a ), except that λ DNA was used. c Cleavage control by reaction time. E. coli DNA was digested at 65 °C at times ranging from 7.5 min to 16 h. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction conducted for 7.5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 4 h; lane 7, 8 h; lane 8, 16 h. d The same as ( c ), except that λ DNA was used. e Cleavage control by the enzyme amount. E. coli DNA was digested at 65 °C with TthHB27I. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, DNA digested with 8 U TthHB27I; lane 2, 4 U; lane 3, 2 U; lane 4, 1 U; lane 5, 0.5 U; lane 6, 0.25 U; lane 7, 0.12 U; lane 8, 0.06 U. f The same as ( e ), except that λ DNA was used
Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).
Techniques: Activity Assay, Incubation