Xba I

Descrição detalhada : Xba I recognizes the sequence T↓*CTAGA and generates fragments with 5'-cohesive termini. The enzyme needs at least two nucleotides around the target sequence before it will cut the cleavage site.Compatible endsXba I ends are compatible with fragments generated by Avr I, Nhe I, and Spe I.IsoschizomersThe enzyme is not known to have isoschizomers.Methylation sensitivityXba I digestion of DNA is inhibited by the dam gene product of E. coli, which methylates the 6N position of adenine in the sequence GATC. The enzyme is also inhibited by 5-methylcytosine or 5-hydroxymethylcytosine at the site (*) indicated on the recognition sequence.Activity in SuRE/Cut Buffer SystemBuffer printed in bold face type is the buffer recommended for optimal activity:A B L M H100% 75-100% 75-100% 75-100% 100%Relative activity in complete PCR mixRelative activity in PCR mix (Taq DNA Polymerase buffer) is 60%. The PCR mix contained ?DNA, primers, 10 mM Tris-HCl (pH 8.3, +20C), 50 mM KCl, 1.5 mM MgCl2, 200 ?M dNTPs, 2.5 U Taq DNA polymerase. The mix was subjected to 25 amplification cycles. Activity in reaction buffer of Pwo SuperYield DNA Polymerase PCR Mix is 25%. When supplemented with GC-RICH Solution, activity increases to 100%.Incubation temperature+37CNumber of cleavage sites on different DNAslambda Ad2 SV40 ?X174 M13mp7 M13mp18 pBR322 pBR328 pUC181 5 0 0 0 1 0 0 1PFGE testedXba I has been tested in Pulsed-Field Gel Electrophoresis (on bacterial chromosomes). For cleavage of genomic DNA (E. coli C 600) embedded in agarose for PFGE analysis, we recommend 10 U enzyme per ?g DNA and approximately 4 hour incubation time.Ligation and recutting assayXba I fragments obtained by complete digestion of 1 ?g pUC18 DNA are ligated with 1 U T4 DNA Ligase in a volume of 10 ?l by incubation for 16 hours at +4C in 66 mM Tris-HCl, 5 mM MgCl2, 5 mM Dithiothreitol, 1 mM ATP, pH 7.5 (at +20C) resulting in >90% recovery of pUC18 DNA.Subsequent re-cutting with Xba I yields >95% of the typical pattern of pUC18 x Xba I fragments.
Sinónimos : restriction enzyme
Armazenamento : -20C
Embalagem : 1X1EA