1/11/2024 0 Comments Noti hfNote: For methylation sensitivity, refer to product specifications. Restriction fragment length polymorphism (RFLP).HF enzymes also exhibit dramatically reduced star activity. High Fidelity (HF) Restriction Enzymes have 100 activity in rCutSmart Buffer single-buffer simplicity means more straightforward and streamlined sample processing. Wide selection of restriction endonuclease specificities NotI has a High Fidelity version NotI-HF ( NEB R3189 ). For all questions regarding availability please call: 0800/BIOLABS (2465227 / german customers) or 00800/24652277 (austrian customers) NotI-HF, Article Number: R3189.Includes universal Tango buffer for double-digestions.NEB has introduced a line of High-Fidelity (HF®) enzymes that provide added flexibility to reaction setup. In general, we recommend 510 units of enzyme per µg DNA, and 1020 units for genomic DNA in a 1 hour digest. Convenient color-coded Five Buffer System NotI has a High Fidelity version NotI-HF ® ( NEB R3189 ). Follow with a quick ('touch') spin-down in a microcentrifuge.Superior quality-stringent quality control and industry leading manufacturing process.NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction. We are excited to announce that all reaction buffers are now BSA-free. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations. SpeI-HF has been reformulated, and now also includes Recombinant Albumin (rAlbumin), beginning with Lot 10192521. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. In addition, the universal Tango buffer is provided for convenience in double digestions. Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. Note: Also available as a FastDigest enzyme for rapid DNA digestion. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes. The phages were precipitated and purified by the double-polyethylene glycol (PEG) precipitation method described above.įull paper Login or join for free to view the full paper.Thermo Scientific NotI restriction enzyme recognizes GC^GGCCGC sites and cuts best at 37☌ in O buffer (Isoschizomers: CciNI). To produce chimeric phages, a colony containing the chimeric phage genome was used to inoculate a liquid culture and shaken overnight at 37 ☌. The inserted gene in the phage vector was amplified by PCR (forward primer: 5′- TTTGGAGCCTTTTTTTTGGAGATTTTCAAC-3′ reverse primer: 5′- CACCACCAGAGCCTGC-3′ PCR conditions: 95 ☌ for 3 min, then 11 cycles of 95 ☌ for 30 s, 52.3 ☌ for 30 s and 72 ☌ for 60 s) and Sanger sequenced (UC Berkeley core facility) to confirm the sequence of the chimeric phage genome. The recombinant plasmid was isolated using a QIAprep Spin Miniprep Kit. A single colony was selected and cultured in LB media with kanamycin (50 μg mL–1) and IPTG (0.1 M) in a shaking incubator at 37 ☌ overnight. The recombinant plasmid was then transformed into Mix and Go competent cells, which were plated on LB with kanamycin (50 μg mL–1) and IPTG (0.1 M) and incubated at 37 ☌ overnight. The g3p-N homologue was ligated into the M13-NotI-Kan vector using T4 DNA ligase. The desired products were isolated by gel electrophoresis and purified with QIAquick Gel Extraction Kit. The M13-NotI-Kan phage vector, in which a NotI restriction site was introduced between the N- and C-terminal domains of M13, was prepared previously.12 The extracted plasmid and M13-NotI-Kan vector were digested by KpnI-HF and NotI-HF. The cells were grown in LB media with ampicillin (10 μg mL–1), and the plasmid was isolated using the QIAprep Spin Miniprep Kit. ![]() A plasmid containing the g3p-N homologue of the source phage flanked by KpnI and NotI restriction sites (see the Supporting Information) was synthesized (IDT) and transformed into Mix and Go competent E. The RBP of M13 (g3p) was engineered to replace its N-terminal domain (g3p-N) with the homologous domain from a phage having specificity toward the target bacterial species (Table 1).
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