Protocol Set up the following reaction in a microcentrifuge tube on ice. NEB Restriction Enzyme Double Digest Protocol Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using. email or call 1-800-NEB-LABS. Set up reaction according to recommended protocol. Having supplied restriction enzymes to the research community for over 45 years, NEB has earned the reputation of being the leader in enzyme technologies. This is the Sequential Double Digest Protocol with Standard Restriction Enzymes. In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest. Are you doing COVID-19 related research? Please note that NEBcloner will also provide detailed double digest protocols using this enzyme. One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 DNA (dammethylated) in 1 hour at 37°C in a total reaction volume of 50 µl. Your profile has been mapped to an Institution, please sign back for your profile updates to be completed. Beginning April 2021, we will be gradually transitioning to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. To save your cart and view previous orders, sign in to your NEB account. EcoRI-HF® We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. This includes personalizing content and advertising. email us, or call 1-800-632-7799. For complete digestion of 1 μg of plasmid DNA please follow our recommended digestion protocol. Please note that NEBcloner will also provide detailed double digest protocols using this enzyme. If star activity is a concern, consider using one of our High Fidelity (HF®) enzymes. It calls for one microgram of your target DNA, five microliters of the restriction buffer, five to 10 units of enzyme, and then supplementing the rest of the 50 microliters with distilled water. Working continuously to be worthy of that distinction, NEB strives to develop enzymes of the highest purity and unparalleled quality. Item# Description List Price Web Price Qty; T1010L: Monarch Plasmid Miniprep Kit - 250 preps: $422.00: $379.80 ADD TO CART: T1010S : Monarch Plasmid Miniprep Kit - 50 preps: $102.00: $91.80 ADD TO CART: T1020L: Monarch DNA Gel Extraction Kit - 250 preps: $594.00: $534.60 ADD TO CART: T1020S: … Take advantage of free shipping for any order totaling over $350. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. Since CutSmart Buffer includes BSA, there are fewer tubes and pipetting steps to worry about. (T4 DNA Ligase should be added last. Time-Saver™ qualified for digestion in 5-15 minutes DNA Sequences and Maps Tool. After the first digestion, the protocol provides you with information on how to adjust the salt concentration so that the second digest can be carried out without a DNA cleanup and buffer exchange step. Setting up a Double Digestion Double digests with NEB's restriction enzymes can be set up in CutSmart Buffer. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid … These enzymes will digest 1 µg of substrate DNA in 5-15 minutes using 1 µl of enzyme under recommended reaction conditions, and can also be used safely in overnight digestions. RESTRICTION ENZYMES FROM NEB Simplify Reaction Setup and Double Digestion with CutSmart® Buffer Over 210 restriction enzymes are 100% active in a single buffer, CutSmart Buffer, making it significantly easier to set up your double digest reactions. ccdB, sacB) in the cloning site to avoid the need for gel purification. Contact your local US Sales Representative. NEBcloner®. To learn more and manage cookies, please refer to our Cookie Statement. Restriction Enzyme Digest Protocol: Cutting Close to DNA End Contact our Customer Service Team by DNA 0.5-1ug . DNA Modifying Enzymes & Cloning Technologies, DNA Assembly, Cloning and Mutagenesis Kits, Protein Expression & Purification Technologies, Optimizing Restriction Endonuclease Reactions. ddwater rest of the volume . Protocol for double digestion (20μl system) Pipette the following into a 0.2ml microfuge tube: Enzyme A 1μl . NEB has introduced a line of High-Fidelity (HF®) enzymes that provide added flexibility to reaction setup. This includes personalizing content and advertising. Last Upload: Oct. 11th, 2016 Working continuously to be worthy of that distinction, NEB strives to develop enzyme of the highest purity and unparalleled quality. Also, find other relevant tools and resources to enable protocol optimization. Restriction Enzyme Digestion (Protocol for NEB CutSmart® Buffer) Kits are available for total RNA purification, plasmid miniprep, gel extraction, and DNA & RNA cleanup. This method of preparation provides DNA fragments of the desired size with … You have been idle for more than 20 minutes, for your security you have been logged out. Learn more and request a sample! Working continuously to be worthy of that distinction, NEB strives to develop enzymes of the highest purity and unparalleled quality. The Nucleoside Digestion Mix is a mixture of enzymes that provides a convenient one-step method to generate single nucleosides from DNA or RNA. Having supplied restriction enzymes to the research community for over 45 years, NEB has earned the reputation of being the leader in enzyme technologies. Having supplied restriction enzymes to the research community for over 45 years, NEB has earned the reputation of being the leader in enzyme technologies. Otherwise, choose an NEBuffer that... Set up reaction according to recommended protocol. For a list of many commonly used restriction enzymes, visit NEB. >195 restriction enzymes are Time-Saver qualified, meaning you can digest DNA in 5-15 minutes, or digest DNA safely overnight. Our latest RUO kit, the Luna® SARS-CoV-2 RT-qPCR Multiplex Assay Kit, enables high throughput workflows for real-time detection of SARS-CoV-2 nucleic acid using hydrolysis probes. If you don't see your country above, please visit our This enzyme has shown to have lower activity on some supercoiled plasmids, with more than 1 unit required to digest 1 μg plasmid DNA. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site. We use cookies to understand how you use our site and to improve the overall user experience. All information on the website has been updated to reflect this change. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site. To learn more and manage cookies, please refer to our Cookie Statement. Please sign back in to continue your session. Our latest RUO kit, the Luna® SARS-CoV-2 RT-qPCR Multiplex Assay Kit, enables high throughput workflows for real-time detection of SARS-CoV-2 nucleic acid using hydrolysis probes. Setting up a Double Digestion. Methylation-sensitive restriction enzyme; Time-Saver™ qualified for digestion in 5-15 minutes; 100% activity in rCutSmart ™ Buffer (over 215 enzymes are available in the same buffer) simplifying double digests Please sign back in to continue your session. We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. Contact your local subsidiary or distributor. Save time and money by placing an order with NEB. Beginning April 2021, we will be gradually transitioning to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Your profile has been mapped to an Institution, please sign back for your profile updates to be completed. Enzyme B μl. Protocols. Restriction Digest Protocol. Learn about our tools that are helping researchers develop diagnostics and vaccines for the SARS-CoV-2 virus. All of NEB's Restriction enzymes have transitioned to a new buffer system. All information on the website has been updated to reflect this change. For simple, visual assay results, the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit includes a color-changing pH indicator for detection of SARS-CoV-2 nucleic acid amplification. The protocol recommends that the first digestion be carried out with the enzyme buffer combination requiring the lowest amount of salt. If your enzyme is a Time-Saver qualified enzyme, it will only require a 5 to 15 minute incubation period. Double digestions can save you time, and this video can offer tips for how to achieve the best results. If you don't see your country above, please visit our Choose a DNA, RNA, genome editing, qPCR calculator from NEB, a leader in production and supply of reagents for the life science industry. Optimizing Restriction Endonuclease Reactions; Restriction Digest Protocol ; Double Digest Protocol with Standard Restriction Enzymes; Usage Guidelines. Having supplied restriction enzymes to the research community for over 45 years, NEB has earned the reputation of being the leader in enzyme technologies. international site. For simple, visual assay results, the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit includes a color-changing pH indicator for detection of SARS-CoV-2 nucleic acid amplification. DNA Assembly, Cloning and Mutagenesis Kits, Protein Expression & Purification Technologies, Optimizing Restriction Endonuclease Reactions. Working continuously to be worthy of that distinction, NEB strives to develop enzyme of the highest purity and unparalleled quality. star activity may result from extended digestion. Monarch Nucleic Acid Purification Kits are optimized for maximum performance and minimal environmental impact. Time-Saver™ qualified for digestion in 5-15 minutes Note that the table... Gently mix the reaction by pipetting up and down and microfuge briefly. BsaI-HF ® v2 . Optimized for quantitative analysis by liquid chromatography-mass spectrometry (LC-MS), this reagent eliminates the need for sequential multi-step, time-consuming digestion protocols. international site. Digest the Destination Plasmid with EcoRI-HF® and PstI: The Destination Plasmid DNA should either be prepared with PCR or contain a toxic gene (e.g. KpnI We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. Visit NEBCutSmart.com for further details. A specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool, NEBcloner . Place your order before 7:30pm EST for overnight delivery. A standard reaction is 50 microliters. This is the Double Digest Protocol with Standard Restriction Enzymes, using a common reaction and same incubation temperature for both enzymes. Learn about our tools that are helping researchers develop diagnostics and vaccines for the SARS-CoV-2 virus. Most often, a serial dilution of the selected restriction enzyme(s) is used to digest the starting material and the desired insert size range is isolated by electrophoresis followed by gel extraction of the DNA. The desired insert size for the clone library determines which enzymes are selected, as well as the digestion conditions. A specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool, NEBcloner . Blocked by overlapping dam methylation and CpG methylation. We use cookies to understand how you use our site and to improve the overall user experience. Beginning April 2021, we will be gradually transitioning to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Restriction enzyme digestion is commonly used in molecular cloning techniques, such as PCR or restriction cloning. Some restriction enzymes require more … You have been idle for more than 20 minutes, for your security you have been logged out. Fill out our Technical Support Form, Web pricing is applicable only to orders placed online at www.neb.ca. Working continuously to be worthy of that distinction, NEB strives to develop enzymes of the highest purity and unparalleled quality. Contact your local subsidiary or distributor. Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes. Otherwise, choose an NEBuffer that results in the most activity for both enzymes. Protocols, Manuals & Usage. All information on the website has been updated to reflect this change. Download a PDF containing pricing for our full product list. For complete digestion of 1 μg of plasmid DNA please follow our recommended digestion protocol. Use NEBcloner to find the right products and protocols for each in your traditional cloning workflow, including double digestion buffers. 1 10 buffer 2μl. Use NEBcloner to find the right products and protocols for each in your traditional cloning workflow, including double digestion buffers. Restriction Digest Protocol. Time-Saver™ qualified for digestion in 5-15 minutes X Companion Products. Are you doing COVID-19 related research? Links to this resource Product Categories: Use this tool to find the right products and protocols for each step (digestion, end modification, ligation and transformation) of your next traditional cloning experiment. However, to speed up the screening process, choose one of NEB's enzymes that are Time-Saver qualified. For maximum convenience and value, columns and buffers are also available separately. It is also used to quickly check the identity of a plasmid by diagnostic digest. To save your cart and view previous orders, sign in to your NEB account. Double digests with NEB's restriction enzymes can be set up in CutSmart Buffer. Convenience >215 restriction enzymes are 100% active in a single buffer – CutSmart™ Buffer.
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