Dna molecules, the only reaction catalysed by the ecori restriction endonuclease was the hydrolysis of the. Autoclaving glassware filled 34 with ddh2o to remove most detergent residue 2. Fastdigest enzymes are an advanced line of fast restriction enzymes that are all 100% active in the universal fastdigest and fastdigest green reaction buffers. This diffusion rate is about 2000fold slower than the diffusion of free protein in solution.
Promega manufacturing and delivery systems continue to be fully operational during the covid19 outbreak. To yield hemagglutinin hatagged murine epor pmowshamepor, an ecori bamhi fragment from the vector pmxeporhairesgfp provided by s. Reprogramming of human fibroblasts to induced pluripotent. For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page. This diffusion rate is about 2000fold slower than the diffusion of the free protein in solution. Aav helper free system, the rep and cap genes have been removed from the viral vector that contains aav2 itrs and are supplied in trans on the plasmid paavrc.
This volume of the enzyme is recommended for preparations of standard concentrations 10 ul, whereas hc enzymes 50 ul should be. Chimeric dna cdna recombinant fp amplicons were purified with a qiaquick purification kit for purification of 100 bp to 10 kb pcr products qiagen. A factor of 4050 can be accounted for by a rotational friction resulting from following the helical path of the dna backbone. Anza restriction enzymes show complete digestion in 15 minutes with no star activity after overnight digestion.
The open reading frame was amplified by pcr from human genomic dna. To request the quick reference card qrc or for additional information, contact. Reaction mixture included 1 g of dna and 1 l of restriction enzyme to a total volume of 20 l, following the. The invitrogen anza restriction enzyme cloning system is a complete system, comprised of. Flow chart describing the general steps of cloning strategy of the tinman gene. Supporting online material materials and methods som text figs. Cloning and characterization of three fatty alcohol oxidase. L invitrogen anza 11 ecori ca dz restriction enzymes. Negative regulation of the ecori restriction enzyme gene is associated with intragenic reverse promoters article pdf available in journal of bacteriology 18919. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and. Media and buffers in detergent free glassware and cultures grown up in detergent free glassware b. This product was subcloned into the invitrogen pentr1a entry vector and an expression plasmid generated using the gateway system to transfer the coding sequences into the pdest 8 destination vector for use in bev system.
Anza restriction enzymes utilize a single buffer and protocol complete. To ensure consistent enzyme performance, thermo scientific restriction enzyme buffers contain bsa, which enhances the stability of many enzymes a. Active protein concentrations for the individual binding experiments were determined by direct titration of the ecori with the 360 bp dna fragment under conditions of stoichiometric binding as described previously 10. Reaction mixture followed recommended protocol, which includes 1 g of dna and 1 l of restriction enzyme in a final volume of 20 l. Our teams are in regular contact with suppliers and distributors worldwide to manage inventory of raw materials to ensure continued availability. Contains one nonsilent mutation at nt632 ga resulting in an change of arg to his. Nuclease free water as required to make up final reaction volume anza 10x buffer or. Supplemental data limp2 is a receptor for lysosomal mannose. The pgem t and pgem t easy vector systems are supplied with 2x rapid ligation buffer. Also available as a fastdigest enzyme for rapid dna digestion. Thermo scientific ecori restriction enzyme recognizes gaattc sites and cuts best at 37c in its own unique buffer. Details for sample preparation and electrophoresis conditions are included in this guide.
Ecori is able to diffuse 150 base pairs on average along dna in 1 second. This is an enzyme that breaks down lipids in the skin, and if an inhibitor could be developed, it might lead to a therapy for acne. A comparative study on the recovery of ecori endonuclease from. Deoxyribonuclease i, amplification grade dnase i, amp grade digests singleand doublestranded dna to oligodeoxyribonucleotides containing a 5. We calculate a diffusion coefficient for ecori of 3. Cloning, expression and functional characterization of the.
Dna invitrogen, were included in all the pcr batches. The invitrogen anza restriction enzyme cloning system utilizes a single buffer system for complete digestions and incorporates anza cloning enzymes into a clear and simple system. Simple cloning exercise in the genome sequence of propionibacterium acnes bacterium that causes acne, you have discovered a gene predicted to encode a lipase. Thermo scientific 10x buffer ecori is the optimal buffer recommended for use with ecori restriction enzyme and is premixed with bsa for enhanced stability. The reaction was initiated by the addition of 5,ul of ecori restriction endonuclease 40 units. These restriction endonucleases have been widely used for gene cloning and mapping, for investigations on gene expression in both prokaryotic and eukaryotic systems, and as. Jan 15, 2010 ecori is able to diffuse 150 base pairs on average along dna in 1 second. An ecorixhoi fragment containing the complete murine adamts cdna was then subcloned into.
After treatment with turbodna free reagent applied biosystems, warrington, uk, cdna was generated using the superscript iii method, with random hexamers invitrogen. Escherichia coli ri ecori dna restriction and modifica. Insert restriction enzyme cuttingfree cloning strategy for expression. To investigate whether plasmid size and the presence of the rop gene are involved in. Make sure that the dna preparation is free of organic solvents and contaminating salts. The extent of protection by ecori methyltransferase is determined by the addition of 40 l nebuffer 2 supplemented with 10 mm mgcl 2 and 5 units of ecori restriction endonuclease. Anza restriction enzymes thermo fisher scientific ca.
Diffusion of the restriction nuclease ecori along dna. S1 to s16 references materials and methods plasmids and reagents. Selection program that can be accessed after a free registration. For more information about lipofectamine 2000 and other transfection reagents, refer to our web site. Nuclease free water is ideal for the preparation of reagents and for use in enzymatic reactions. No toxic agents, such as depc, are used in the production of this water, so as to avoid inhibition in enzymatic reactions. These restriction endonucleases have been widely used for gene cloning and mapping, for investigations on gene expression in both prokaryotic and eukaryotic systems, and as models for the study of proteindna interactions. Both kpni and ecori amplified fp cdna fragments gfpuv, amcyan, zsgreen. Ecori endonuclease journal of biological chemistry. After treatment with turbodna free reagent applied biosystems, warrington, uk, cdna was generated using the superscript iii method, with. Incubation at 37c for 30 minutes is followed by analysis on an agarose gel. Dec, 2011 the binding of ecor1 to a 90bp dna duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label free with timeresolved second harmonic sh spectroscopy. Strr enda1 nupg invitrogen was used as the host strain for construction of all of the plasmids. Ecori, noti, and bamhi from multiple suppliers were tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3overhang or 5 overhang containing oligonucleotides.
Ecor i from escherichia coli bs5 multiple sizes available sigma. Quality control tests indicated enzyme preparations free of contaminants. The binding of ecor1 to a 90bp dna duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label free with timeresolved second harmonic sh spectroscopy. Plasmid dna was obtained from cultures with the qiaprep miniprep kit qiagen and analyzed for the presence of the insert by cutting the plasmid with ecori. Although the ecori restriction enzyme has been well characterized, regulation of its. The incubation period may be extended to increase the number of colonies after transformation. Inheritance of lowfrequency regulatory snps and a rare null. Ligation reactions using this buffer may be incubated for 1 hour at room temperature. Embryonic stem cellsinduced pluripotent stem cells reprogramming of human fibroblasts to induced pluripotent stem cells under xeno free conditions ignasi rodri. Simplify traditional restriction enzyme cloning with the invitrogen anza restriction enzyme cloning system. Preparing glassware and media eliminate detergent 1. Pdf negative regulation of the ecori restriction enzyme. Hf enzymes also exhibit dramatically reduced star activity.
The pcr product was cloned with a topo ta cloning kit invitrogen into top10f strain cells. Escherichia coli ri ecori endonuclease is a wellknown restriction. The map, notes, and annotations on this page and in the sequencemap file are ed material. Negative regulation of the ecori restriction enzyme gene is. Supplemental data limp2 is a receptor for lysosomal. Supercoiled dna generally requires more units of enzyme than linear dna for complete digestion. Anza restriction enzyme cloning system fisher scientific. The binding of ecor1 to the recognition sequence in dna appears as a rapid increase in. The percent degradation is determined by capillary electrophoresis and peak analysis. Check for optimal reaction temperature on the promega product information sheet supplied with the enzyme. Pdf a fundamental step in molecular biology is the cloning of a dna fragment insert into a plasmid vector. Media and buffers in detergent free glassware and cultures grown up in detergent free. Binding and cleavage of dna with the restriction enzyme ecor1.
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