New England Biolabs, M0202L, T4 DNA Ligase

Looking for additional T4 DNA Ligase formulations and variants? Related Categories DNA Ligases Applications BioBrick, ®, Assembly,, Cloning Ligation,, NEBridge® Golden Gate Assembly Specification Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer. Concentration: 400,000 cohesive end units/ml and 2,000,000 cohesive end units/ml Reaction Conditions 1X T4 DNA Ligase Reaction Buffer Incubate at 16°C 1X T4 DNA Ligase Reaction Buffer 50 mM Tris-HCl 10 mM MgCl2 1 mM ATP 10 mM DTT (pH 7.5 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 10 minutes FAQ Q: What are some potential problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure? A: * Ligation failed because there was no ATP or Mg2+. Use the supplied buffer or add ATP to a compatible buffer. The ATP in buffers older than one year may have degraded enough to cause problems. When supplementing with ATP, be sure to use ribo ATP as deoxyribo ATP will not work. * Ligation failed due to high salt or EDTA in the reaction. Clean up the DNA. * CIP, BAP or SAP not completely inactivated from dephosphorylation step. Follow the recommended procedure to remove the phosphatase. * Ligation produced only linear DNA because the DNA concentration was too high. Keep the total DNA concentration between 1-10 μg/ml. * The ligated end was a single base overhang. Use up to 5 μl concentrated ligase at 16°C overnight. * The insert and the plasmid do not have phosphates. Note: primers may not have phosphates leading to problems blunt end ligating into CIPed vectors. Order primers with phosphates or kinase the primers. * Too much ligation mixture was added to the cells. Add between 1-5 μl to 50 μl competent cells. * The insert was large and the conditions didn't allow circularization. Reduce the insert concentration and use concentrated ligase at 16°C overnight. * The ligase was inactive. Test on lambda HindIII or other convenient substrate. * The ligation mix contained PEG and was incubated overnight. Extended ligation with PEG causes a drop off in transformation efficiency. This could be due to the gradual production of large linear pieces of DNA that can inhibit transformation. The buffer for the Quick Ligation Kit (NEB# M2200) contains PEG. * The ligation mix was not purified prior to electroporation. The buffer must be removed or a spark will be generated by the salt. Dialyse the sample or use a spin column to purify. The PEG in the Quick Ligation Kit Buffer (NEB# M2200 ) prevents sparking but it also prevents electroporation. PEG must be removed using a spin column. Q: What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation to fail? A: * The restriction enzyme did not cleave efficiently. If cleaving near the end of a PCR * fragment leave at least 6 bases past the restriction site. Test the restriction enzyme on a control substrate. * The restriction enzyme was not completely inactivated. Phenol/ETOH purify the DNA if the enzyme cannot be heat inactivated. * Star activity from the restriction digest cleaved the vector or insert. Check the DNA on a gel. If there is an extra band, reduce the amount of enzyme or time for the restriction digest. * The DNA or restriction enzyme contained exonuclease or phosphatase that damaged the ends. Phenol/ETOH purify the DNA. Check the enzyme QC data and notes. If the ligation QC is poor or the exonuclease level is high reduce the amount of enzyme or incubation time. * The PCR process is covered by patents owned by Roche Molecular Systems, Inc. Q: What controls should be run to test the cells and DNA when using T4 DNA Ligase? A: Note: Use the same DNA concentration for each control, typically 0.1 -1.0 ng per transformation. 1. Transforming uncut vector in the competent cells checks cell viability and tests the antibiotic resistance of the plasmid. Plate on media and media plus antibiotic. 2. Linearized vector tests for background due to uncleaved plasmid. Should be <1% of the value obtained in 1. 3. Cut and religated plasmid tests ligase activity and DNA end integrity. Should be near the value obtained in 1. 4. Cut, phosphatased, religated plasmid tests background due to incomplete phosphatase treatment. Should be <1% of the value obtained in 1. Q: When should T4 DNA Ligase be the enzyme of choice? A: T4 DNA Ligase should be used to ligate cohesive ends (10 minutes at room temperature) or blunt ends (2 hours at room temperature) or if the ligation is to be done overnight. T4 DNA Ligase should be used if heat inactivation is required. Concentrated T4 DNA Ligase is suggested for ligating single base overhangs or for large constructs which require longer incubation times to circularize, such as BAC (Bacterial Artificial Chromosome) construction. Q: Can the T4 DNA Ligase be used with the Quick Ligase buffer? A: Yes, concentrated T4 DNA Ligase can be substituted directly. When using regular T4 DNA Ligase, increase the reaction time to 15 minutes from 5 minutes. Do not heat kill the reaction because heat treating the PEG in the Quick Ligase reaction buffer will inhibit transformation. Transformation efficiency will also decrease if the reaction time is extended. Q: What is the definition of a Weiss Unit and a Cohesive End Unit? A: A Weiss unit is based on the T4 DNA ligase catalyzed ATP-PPi exchange reaction as described by Bernard Weiss, Charles Richardson and colleagues (Weiss, B., et. al., (1968) J. Biol. Chem., 243, 4556). One Weiss unit is defined as the amount of enzyme required to convert 1 nmol of 32P-labeled inorganic pyrophosphate into Norit adsorbable material in 20 minutes at 37°C, using specified reaction conditions (Weiss, B., et al., (1968) J. Biol. Chem., 243, 4543). Norit is a type of activated carbon. A Cohesive End Unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA (5´ DNA termini concentration of 0.12 µM (300 µg/ml)) in 20 µl of 1X T4 DNA Ligase Buffer in 30 minutes at 16°C. Q: What is the difference between the two definitions and why does NEB use the Cohesive End Unit? A: The Weiss unit is not a direct measurement of DNA ligation; it is an indirect measurement of the enzyme adenylation reaction, followed by determining ATP-PPi exchange. NEB did not think that the Weiss unit was informative enough, because the most prevalent use of T4 DNA ligase is for the ligation of DNA. As the Cohesive End Unit involves the direct measurement of DNA ligation, NEB feels that it more practically relevant. Q: How much DNA should be used in a ligation using T4 DNA Ligase? A: The unit definition uses 0.12 μM (300 μg/ml) lambda HindIII fragments. The high DNA concentration can be used for linker ligation. To promote circle formation, useful in transformation, a lower total DNA concentration should be used. The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Vector:Insert molar ratios between 1:1 and 1:10 are recommended (1:3 is typical). If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. Q: Can T4 DNA Ligase be used in other NEBuffers, including rCutSmart? A: Ligation can also be performed in any of the standard restriction endonuclease NEBuffers, including rCutSmart® Buffer, or in T4 Polynucleotide Kinase Buffer if supplemented with 1 mM ATP. Please be sure to use riboATP (NEB #P0756) as deoxyriboATP will not work. When using NEBuffer 3 or r3.1, high levels of salt in the DNA preparation may decrease ligation efficiency. To see its % functional activity in rCutsmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. Q: Can T4 DNA Ligase be heat inactivated? A: Yes, heat at 65 °C for 10 minutes. Do not heat inactivate if there is PEG in the reaction buffer (Quick Ligation Kit buffer NEB# M2200 ) because transformation will be inhibited.