Formula. DNA calculations to convert µg to pmol for double-stranded and single-stranded DNA, convert micrograms of DNA to pmol ends, calculate vector:insert molar ratio and convert OD260 readings to µg/ml. Also calculate molarity of solutions, perform molar conversions, calculate dilutions and perform other calculations common in molecular ...Driving calculator. Travelmath provides driving information to help you plan a road trip. You can measure the driving distance between two cities based on actual turn-by-turn directions. Or figure out the driving time to see if you need to stop overnight at a hotel or if you can drive straight through. To stay within your budget, make sure you calculate the cost of driving based on your car's ...10 μl of 2X Gibson Assembly Master Mix was incubated with 6 fragments (5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmol each) in a final volume of 20 μl at 50°C for 60 minutes. NEB 5-alpha Competent E. coli (NEB #C2987) were transformed with 2 μl of the master mix/fragment mixture using the transformation protocol.Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix. Heat shock at 42°C for 30 seconds.*. Do not mix. Transfer tubes on ice for 2 minutes. Add 950 μl of room temperature SOC media* to tubes. Place the tube at 37°C for 60 minutes.FAQ: Can longer or shorter incubation times be used? Yes. For assembling 2-3 fragments, 15 minute incubation times are sufficient. For assembling 4-6 fragments, 60 minute incubation times are recommended. Reaction times less than 15 minutes are generally not recommended. Extended incubation times (up to 4 hours) have been …NEBuilder® HiFi DNA Assembly, developed by New England Biolabs, is an effective method for the high-fidelity assembly of multiple DNA fragments. Advantages over Gibson Assembly Master Mix include higher efficiency, higher accuracy, the ability to assemble both 5´- and 3´-restriction enzyme mismatches, the ability to bridge two double ...Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products. Select the product group of the polymerase or kit you plan to use. Select the polymerase or kit from the list of products. If needed, modify the recommended primer concentration. Enter primer sequences (with up to 3 ambiguous bases).NEB offers a variety of interactive tools to help you accurately design primers for your specific needs. ... NEBuilder Assembly Tool can be used to design primers for your NEBuilder HiFi or Gibson Assembly reactions based on entered fragment sequences and the polymerase being used for amplification. This video will highlight some useful updates ...Ipswich, MA (December 12th, 2012) – New England Biolabs, Inc. (NEB) today released the Gibson Assembly Cloning Kit and online primer design tool, NEBuilder™ , for the cloning of DNA fragments using the Gibson Assembly approach. This kit includes NEB's robust Gibson Assembly Master Mix and NEB 5-alpha competent .Mileage Calculator. Use the following mileage calculator to determine the travel distance, in terms of miles, and time taken by car to travel between two locations in the United States, disregarding traffic conditions. From: To: This mileage calculator estimates the number of driving miles between two locations in the United States.Access protocols related to NEB products. Find protocols. Selection Tools. Get help with selecting an NEB product for your application. Browse selection charts. Troubleshooting Guides. Find the help you need in our extensive troubleshooting guides. Browse troubleshooting guides. Usage Guidelines & TipsThe molar ratio of insert:vector is set to 2:1, as recommend by our In-Fusion Cloning protocol. The molar ratio stays the same with multiple inserts. For example, the molar ratio of two inserts with one vector should be 2:2:1. The total DNA amount (insert + vector) provided by the calculator is 200 ng, which is optimal for a 10-µl In-Fusion ...GPA Calculator is a simple tool built with the mail goal to help +2 students estimate and calculate their GPAs accurately. Calculate your +2 GPA effortlessly with our NEB-compliant tool. Stay on track, monitor your academic progress, and set new goals. Achieve academic excellence with our user-friendly GPA Calculator. Start calculating your GPA ...Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products. Select the product group of the polymerase or kit you plan to use. Select the polymerase or kit from the list of products. If needed, modify the recommended primer concentration. Enter primer sequences (with up to 3 ambiguous bases).DNA assembly refers to a molecular cloning method that physically links together multiple fragments of DNA, in an end-to-end fashion, to achieve a designed, higher-order assembly prior to joining to a vector. This process is the cornerstone of the synthetic biology field and allows the construction of novel biological systems and devices using ...2(1.5 mM at 1X dilution). HF buffer is recommended as the default buffer for high-fidelity amplification. Phusion GC Buffer (NEB #B0519)1.5/1.5 ml (5X) 5X Phusion GC Buffer contains 7.5 mM MgCl. 2(1.5 mM at 1X dilution). For difficult templates, such as GC-rich templates or those with secondary structure, GC buffer can improve enzyme performance.This is part of the #BlackCardioInHistory series. #AHAEarlyCareerVoice is partnering with #BlackInCardio to feature a series of profiles of black/African American Cardiologists for...Generally, 25–35 cycles yields sufficient product. When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol is recommended. The PCR products generated using Phusion DNA Polymerase have blunt ends; if cloning is the next step, then blunt-end cloning is recommended.GeoGebra Scientific Calculator is a free online tool that lets you perform calculations with fractions, statistics and exponential functions, logarithms, trigonometry and much more. You can also explore interactive activities and simulations related to various topics in mathematics and science.For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). Heat inactivate at 65°C for 10 minutes. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.For Gibson Master Mix, use 3.75uL in a 5uL total volume (or 7.5uL in a 10uL total volume). Calculating how much DNA to add to your Gibson Reaction: NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. …Figure 1: Not your average DNA assembly reagent. Figure 2: Overview of the NEBuilder HiFi DNA Assembly method. Figure 3: NEBuilder HiFi DNA Assembly offers improved efficiency in 4-fragment assembly reactions. Four fragments (~20 fmol) with 20 bp overlap were assembled using NEBuilder HiFi DNA Assembly Master Mix ( NEB #E2621 ), …This tutorial describes the use of the NEBioCalculator web tool to optimize the molar ratio between vector and insert DNA for use in a ligation reaction.Gibson Assembly, developed by Dr. Daniel Gibson and his colleagues at the J. Craig Vendor Institute is an effective method for the assembly of multiple DNA fragments. This is accomplished in a single tube isothermal reaction with Gibson Assembly Master Mix. The method utilizes adjacent DNA fragments with complementary ends which can be added ...Gibson Assembly™ employs three enzymatic activities in a single-tube reaction: 5´ exonuclease, the 3´ extension activity of a DNA polymerase and DNA ligase activity. The 5´ exonuclease activity chews back the 5´ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed …About New England Biolabs Established in the mid 1970's, New England Biolabs, Inc. is the industry leader in the discovery and production of enzymes for molecular biology applications and now offers the largest selection of recombinant and native enzymes for genomic research. NEB continues to expand its product offerings into areas related to ...How to use the T m calculator. The calculator calculates recommended T m (melting temperature) of primers and PCR annealing temperature based on the primer pair sequence, primer concentration, and DNA polymerase used in PCR. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient.NEBuilder ® HiFi DNA Assembly:. Master Mix NEB #E2621. Cloning Kit NEB #E5520. Bundle for Large Fragments NEB #E2623. NEB Gibson Assembly ®:. Master Mix NEB #E5510. Cloning Kit NEB #E2611. NEBridge ® Golden Gate Assembly:. BsaI-HFv2 Kit NEB #E1601. BsmBI-v2 Kit NEB #E1602 NEBridge ® Ligase Master Mix NEB #M1100. USER™ Enzyme NEB #M5505 Thermolabile USER II Enzyme NEB #M5508 ...Try these video introductions to NEBuilder v2. NEBuilder Assembly Tool 2.0 What's New? Detailed information on features is also available on the Help page. NEBuilder Assembly Tool can be used to design primers for NEBuilder HiFi DNA Assembly or Gibson Assembly reactions.Updated resource links to NEBuilder and Gibson manuals. Minor revisions to About page and NEB Legal disclaimers. v2.2.5 July 8, 2019. Fixed restriction enzyme digest display issue where duplicate sites were shown. Updated Restriction enzyme data files. v2.2.4 June 10, 2019. Added Q5U Hot Start polymerase as a PCR option. v2.2.3 …tools.neb.com Online sequence analysis tools: NEBcutter: REBsites: BLAST against REBASE: REBASE Genomes: Tech Support Feedback NEB Overview Site Map Trademarks Legal ...This is part of the #BlackCardioInHistory series. #AHAEarlyCareerVoice is partnering with #BlackInCardio to feature a series of profiles of black/African American Cardiologists for...Specification: 10 μl of 2X Gibson Assembly Master Mix was incubated with 6 fragments (5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmol each) in a final volume of 20 μl at 50°C for 60 minutes. NEB 5-alpha Competent E. coli (NEB #C2987) were transformed with 2 μl of the master mix/fragment mixture using the ...NEBuilder Assembly Tool can be used to design primers for your NEBuilder HiFi DNA or Gibson Assembly reactions, based on the entered fragment sequences and the polymerase being used for amplification. Basic Usage: Set preferences, including the number of fragments and the PCR enzyme. Change settings at any time and the results will be instantly ...Formula. moles protein (mol) = mass of protein (g)/ (molecular weight (Da)) number of molecules = moles of protein (mol) x 6.022e23 molecules/mol. Choose a DNA, RNA, qPCR calculator from NEB, a leader in production and supply of reagents for the life science industry.Gibson Assembly™ employs three enzymatic activities in a single-tube reaction: 5´ exonuclease, the 3´ extension activity of a DNA polymerase and DNA ligase activity. The 5´ exonuclease activity chews back the 5´ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions.The molar ratio of insert:vector is set to 2:1, as recommend by our In-Fusion Cloning protocol. The molar ratio stays the same with multiple inserts. For example, the molar ratio of two inserts with one vector should be 2:2:1. The total DNA amount (insert + vector) provided by the calculator is 200 ng, which is optimal for a 10-µl In-Fusion ...Browse NEB's Gibson Assembly products for cloning ... Please review and update your order accordingly If you have any questions, please contact Customer Service at [email protected] or 1-800-632-5227 x 8. Continue. To Request Technical Support. Fill out our Technical Support Form, email us, ...No Gibson's have worked thus far when using 1:1 equimolar ratios nor 1:2 backbone : insert ratios when using the NEB Bio Calculator. All Gibson Assembly reactions were ran in the thermocycler at ...There are four state capitals named after U.S. Presidents. They are Jefferson City, Mo., named for Thomas Jefferson; Lincoln, Neb., named for Abraham Lincoln; Jackson, Miss., named...Even if you don’t have a physical calculator at home, there are plenty of resources available online. Here are some of the best online calculators available for a variety of uses, ...NEBioCalculator®. Use this tool for your scientific calculations and conversions for DNA and RNA. Options include conversion of mass to moles, ligation amounts, conversion of OD to concentration, dilution and molarity. Additional features include sgRNA Template Oligo Design and qPCR library quantification.Script. NEBioCalculator is an easy-to use tool that helps with various biomass calculations. The double-stranded DNA, mass-to-ends module is useful when setting up a phosphorylation reaction using T4 Polynucleotide Kinase, also known as T4 PNK, because it converts linear double-stranded DNA mass to moles of DNA ends.Dephosphorylation of DNA 5′-ends using Quick CIP in a Restriction Enzyme Reaction. The phosphatase can be added directly into the digestion reaction during or after DNA digestion. Add 1 μl of Quick CIP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 10 minutes. Quick CIP is active in all NEB restriction ...First, click on the ligation calculator module. Enter 2 kb for your DNA insert length, and 6 kb for your vector DNA length, making sure that the units selected are correct. We recommend 27 femtomoles of the vector to ensure you have enough DNA ends to conduct a successful ligation. Using the NEBioCalculator double-stranded DNA mass to moles ...Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products. Select the product group of the polymerase or kit you plan to use. Select the polymerase or kit from the list of products. If needed, modify the recommended primer concentration. Enter primer sequences (with up to 3 ambiguous bases).Gibson Assembly Master Mix (2X) NEB 5-alpha Competent E. coli (High Efficiency) SOC Outgrowth Medium Positive Controls: Gibson Assembly Positive Control 2 overlapping dsDNA fragments for control assembly pUC19 Control DNA for NEB 5-alpha Competent E. coli Required Materials Not Included: DNA Polymerase (for generating PCR products):The group of cloning methods we refer to as "seamless cloning" typically combine attributes of more established cloning methods to create a unique solution to allow sequence-independent and scarless insertion of one or more fragments of DNA into a plasmid vector. Various commercial systems, such as NEBuilder HiFi DNA Assembly, In-Fusion ®, and ...Gibson Assembly, developed by Dr. Daniel Gibson and his colleagues at the J. Craig Vendor Institute is an effective method for the assembly of multiple DNA fragments. This is accomplished in a single tube isothermal reaction with Gibson Assembly Master Mix. The method utilizes adjacent DNA fragments with complementary ends which can be added ...Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products. NEB Tm Calculator (tmcalculator.neb.com) TmCalculator. version 1.16.5. HELP ABOUT Tech Support Feedback NEB Overview Site Map. Trademarks Legal and ...Gibson Assembly Calculator: ... The original Gibson et al. paper was published in 2009, and showed how DNA segments to be joined were treated with a mixture of a DNA ligase, an 5' exonuclease and a polymerase, a variant of which is now obtainable commercially from New England Biolabs, see here. The exonuclease digests back the 5' region of each ...To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool, NEBioCalculator , or using the following formula: pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons) 50 ng of 5000 bp dsDNA is about 0.015 pmols. 50 ng of 500 bp dsDNA is …We used NEB's DpnI (NEB #R0176) Dpn1 (NEB #R0176) digestion of a PCR reaction selectively destroys the plasmid template, leaving the PCR product intact. Indeed Dpn1 only cleaves E. coli Dam methylase-methylated plasmid DNA, but does not cleave the PCR product, since it is not methylated. 1. For a 20 μl reaction, mix in a tube:A) NEB Stable competent cells or B) Stbl3 competent cells were transformed with 2 µl of a pUC-5xREP Gibson Assembly reaction containing 2.2 ng (0.00125 pmol) pUC19 vector and approximately 1 ng (0.0028 pmol) 5xREP insert.Push the Limits of Golden Gate Assembly. 50+ fragment DNA assembly achievable with high efficiency and accuracy! With constant advances both in the development of new enzymes (e.g., PaqCI ®, BsaI-HF ® v2 and BsmBI-v2) and research on maximizing enzyme functionality (e.g., ligase fidelity), NEB has become the industry leader in pushing the ...We generally recommend using Q5 Hot Start High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of Q5 Hot Start High-Fidelity DNA Polymerase may vary from 10-40 units/ml (0.5-2 units/50 μl reaction) depending on amplicon length and difficulty.FAQ: Can longer or shorter incubation times be used? Yes. For assembling 2-3 fragments, 15 minute incubation times are sufficient. For assembling 4-6 fragments, 60 minute incubation times are recommended. Reaction times less than 15 minutes are generally not recommended. Extended incubation times (up to 4 hours) have been shown to improve ...Figure 1: Overview of the Gibson Assembly Cloning Method Specification 10 µl of 2X Gibson Assembly Master Mix was incubated with 6 fragments (5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmol each) in a final volume of 20 µl at 50°C for 60 minutes. NEB 5-alpha Competent E. coli (NEB #C2987) were transformed with. Options include conversion of mass to moles, ligation amounts, First, click on the ligation calculator module. NEBuilder ® HiFi DNA Assembly:. Master Mix NEB #E2621. Cloning Kit NEB #E5520. Bundle for Large Fragments NEB #E2623. NEB Gibson Assembly ®:. Master Mix NEB #E5510. Cloning Kit NEB #E2611. NEBridge ® Golden Gate Assembly:. BsaI-HFv2 Kit NEB #E1601. BsmBI-v2 Kit NEB #E1602 NEBridge ® Ligase Master Mix NEB #M1100. USER™ Enzyme NEB #M5505 Thermolabile USER II Enzyme NEB #M5508 ...How to use the T m calculator. The calculator calculates recommended T m (melting temperature) of primers and PCR annealing temperature based on the primer pair sequence, primer concentration, and DNA polymerase used in PCR. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient. Daniel G. Gibson, of the J. Craig Venter Institute, d 10 μl of 2X Gibson Assembly Master Mix was incubated with 6 fragments (5 fragments of 400 bp and one of 2,780 bp, with 40 bp overlap, 0.05 pmol each) in a final volume of 20 μl at 50°C for 60 minutes. NEB 5-alpha Competent E. coli (NEB #C2987) were transformed with 2 μl of the master mix/fragment mixture using the transformation protocol. Ipswich, MA (December 12th, 2012) – New Eng...
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