MBI Fermentas
MBI Fermentas
Restriction digestion with fewer reagents in less timeIt's cloning time—you want to excise your insert from a vector with two different enzymes. Looking at the old enzyme poster on the fridge you notice that they don’t work in the same buffer. Great Add an extra hour to your schedule for back to back digestions. Wouldn't it be easier to just have one buffer for all your enzymes? Well it'sa good thing we have a single buffer that works on all 176 Thermo Scientific FastDigest restriction Enzymes!Try our FastDigest enzymes today and experience:100% activity in a universal buffer that is even compatible with our Thermo Scientific modifying enzymesComplete digestions in 5-15 minutesNo star activity*Direct sample loading on gels with FastDigest Green Buffer – no extra loading buffer necessaryTested protocols and recommendations for digestion of plasmid DNA, PCR products, and genomic DNASo, no more panicking for double digestions. With FastDigest you get the pleasure of o Ne buffer for all your enzymes and much more time for other experiments.*In most cases, star activity is the result of long incubation times or too much enzyme in the reaction. Our short, 5-15 minute, incubation times and recommended enzyme amount Alleviates this problem.More InformationCheck out our FastDigest Restriction EnzymesView our Double Digestion video to see how fast and convenient FastDigest enzymes are! For Research Use Only. Not for use in diagnostic procedures.
1. Blue-White Screening Blue-white screening is a widely used technique to examine successful cloning. In this method the insert is cloned into a vector containing a lacΖα sequence encoding the α-peptide, a functional subunit of the β-galactosidase enzyme. The plasmid must be transformed into a special strain of E.coli with the lacΖΔΜ15 mutation. An empty vector will produce blue colonies since the activity of the α-peptide (β-galactosidase) remains intact. The colorless X-gal (lactose analog) provided in the screening plates is hydrolyzed by β-galactosidase to form a blue pigment (5,5'-dibromo-4,4'-dichloro-indigo). If the vector contains the DNA insert Disrupting the lacΖα sequence, then the α-peptide will not be expressed and X-gal will not be hydrolyzed. Thus, the colonies will be white if the DNA insert is present. It is possible to obtain false positives (white colonies with no inser Positive selection vectors Conditionive express is to use a positive selection vector. Positive selection vectors conditionally express a lethal gene, such as a restriction enzyme that digests the Genomic DNA of the bacterial host. The expression of the lethal gene is disrupted by ligation of a DNA insert into the cloning site. As a result, only cells with recombinant plasmids are able to grow. This approach can save time and costs since it typically Diagnostic Restriction DigestRestriction enzyme digests can also be performed to determine the correct insert. First, isolate plasmid DNA from an overnight bacterial culture using a plasmid Miniprep kit such as Thermo Scientific GeneJET Plasmid Miniprep Kit. Our REsearch restriction site mapping tool can be used to ide Restriction restriction restriction enzyme Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Run the digested plasmid on an agarose gel to verify that the vector backbone and insert are of the expected sizes. 4. Colony PCRThe presence of the DNA insert can also be determined by screening bacterial colonies using PCR. The primers may be insert-specific, To determine the orientation of the insert, a combination of vector-specific and insert-specific primers is recommended. Colony screening by PCR is suitable for inserts shorter than 3 kb. Individual colonies can be The PCR portion of the colony may be used to inoculate a culture plat SequencingThe most accurate way to verify the recombinant colonies is by sequencing. The plasmid DNA is first isolated from an overnight bacterial culture. The insert can be identified by Sanger sequencing using sequencing primers For the vector. Sequencing across the entire insert is required to verify the exact sequence of insert.
MBI Fermentas endonuclease/protein/PCR is welcome to purchase.
Endonuclease
MBI Fermentas
Restriction digestion with fewer reagents in less timeIt's cloning time—you want to excise your insert from a vector with two different enzymes. Looking at the old enzyme poster on the fridge you notice that they don’t work in the same buffer. Great Add an extra hour to your schedule for back to back digestions. Wouldn't it be easier to just have one buffer for all your enzymes? Well it'sa good thing we have a single buffer that works on all 176 Thermo Scientific FastDigest restriction Enzymes!Try our FastDigest enzymes today and experience:100% activity in a universal buffer that is even compatible with our Thermo Scientific modifying enzymesComplete digestions in 5-15 minutesNo star activity*Direct sample loading on gels with FastDigest Green Buffer – no extra loading buffer necessaryTested protocols and recommendations for digestion of plasmid DNA, PCR products, and genomic DNASo, no more panicking for double digestions. With FastDigest you get the pleasure of o Ne buffer for all your enzymes and much more time for other experiments.*In most cases, star activity is the result of long incubation times or too much enzyme in the reaction. Our short, 5-15 minute, incubation times and recommended enzyme amount Alleviates this problem.More InformationCheck out our FastDigest Restriction EnzymesView our Double Digestion video to see how fast and convenient FastDigest enzymes are! For Research Use Only. Not for use in diagnostic procedures.
1. Blue-White Screening Blue-white screening is a widely used technique to examine successful cloning. In this method the insert is cloned into a vector containing a lacΖα sequence encoding the α-peptide, a functional subunit of the β-galactosidase enzyme. The plasmid must be transformed into a special strain of E.coli with the lacΖΔΜ15 mutation. An empty vector will produce blue colonies since the activity of the α-peptide (β-galactosidase) remains intact. The colorless X-gal (lactose analog) provided in the screening plates is hydrolyzed by β-galactosidase to form a blue pigment (5,5'-dibromo-4,4'-dichloro-indigo). If the vector contains the DNA insert Disrupting the lacΖα sequence, then the α-peptide will not be expressed and X-gal will not be hydrolyzed. Thus, the colonies will be white if the DNA insert is present. It is possible to obtain false positives (white colonies with no inser Positive selection vectors Conditionive express is to use a positive selection vector. Positive selection vectors conditionally express a lethal gene, such as a restriction enzyme that digests the Genomic DNA of the bacterial host. The expression of the lethal gene is disrupted by ligation of a DNA insert into the cloning site. As a result, only cells with recombinant plasmids are able to grow. This approach can save time and costs since it typically Diagnostic Restriction DigestRestriction enzyme digests can also be performed to determine the correct insert. First, isolate plasmid DNA from an overnight bacterial culture using a plasmid Miniprep kit such as Thermo Scientific GeneJET Plasmid Miniprep Kit. Our REsearch restriction site mapping tool can be used to ide Restriction restriction restriction enzyme Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Choose Run the digested plasmid on an agarose gel to verify that the vector backbone and insert are of the expected sizes. 4. Colony PCRThe presence of the DNA insert can also be determined by screening bacterial colonies using PCR. The primers may be insert-specific, To determine the orientation of the insert, a combination of vector-specific and insert-specific primers is recommended. Colony screening by PCR is suitable for inserts shorter than 3 kb. Individual colonies can be The PCR portion of the colony may be used to inoculate a culture plat SequencingThe most accurate way to verify the recombinant colonies is by sequencing. The plasmid DNA is first isolated from an overnight bacterial culture. The insert can be identified by Sanger sequencing using sequencing primers For the vector. Sequencing across the entire insert is required to verify the exact sequence of insert.
MBI Fermentas endonuclease/protein/PCR is welcome to purchase.
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