New England Biolabs, M0318L, T7 DNA Ligase

T7 DNA Ligase will ligate these substrates: Related Categories DNA Ligases Applications Cloning Ligation Specification Unit Definition One unit is defined as the amount of enzyme required to give 50% ligation of 100 ng HindIII fragments of λ DNA in a total reaction volume of 20 μl in 30 minutes at 25°C in 1X StickTogether DNA Ligase Buffer. Reaction Conditions 1X StickTogether™ DNA Ligase Buffer Incubate at 25°C 1X StickTogether™ DNA Ligase Buffer 66 mM Tris-HCl 10 mM MgCl2 1 mM ATP 1 mM DTT 7.5% Polyethylene glycol (PEG 6000) (pH 7.6 @ 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 No FAQ Q: T7 DNA Ligase is described as an enzyme that only ligates DNA fragments with cohesive ends. What length of cohesive end can T7 DNA Ligase ligate? A: T7 DNA Ligase can ligate DNA fragments with cohesive ends that are 2 bps or longer. T7 DNA Ligase ligates 1 bp overhangs inefficiently. T7 DNA Ligase does not effectively ligate blunt end DNA fragments, except under reaction conditions of very high PEG concentrations (20-30% w/v). Q: Can T7 DNA Ligase be used with a buffer that does not contain PEG? A: Yes. We would recommend the use of 1X T4 DNA Ligase Buffer. Please note that the specific activity of T7 DNA Ligase in a buffer that does not contain PEG is lower by about 10-fold. Q: Can T7 DNA Ligase be heat inactivated? A: Yes, heating a reaction containing T7 DNA Ligase at 65 °C for 10 minutes will inactivate the enzyme. However, the reaction needs to be performed in a buffer without PEG. Do not heat inactivate if there is PEG in the reaction buffer, as transformation will be inhibited. Q: What are some potential problems with the ligation reaction using T7 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 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 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 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 are some other problems that should be considered when trouble shooting a transformation problem? A: The cells are not viable. Run the controls listed below and obtain new cells if needed. The cells are not competent. Run the controls listed below and obtain new cells if needed. The recombinant protein is not well tolerated by E. coli. Try making a fusion with maltose binding protein using the pMAL System (NEB# E8000S). Try another expression system that doesn't involve E. coli. The ligated DNA included inverted and tandem repeats selected against by E. coli. Remove the repeat sequence if possible. Try another expression system that doesn't involve E. coli. The insert DNA was taken from mammal or plant and contains methylated cytosine which is degraded by many E. coli strains. Use a strain deficient in mcrA, mcrBC and mrr. Construct is too large (>10,000 bp) for transformation into chemically competent cells. Use electroporation. Q: What problems can be encountered in the restriction digest that can cause ligation using T7 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. he 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: How much DNA should be used in a ligation using T7 DNA Ligase? A: To promote circle formation, useful in transformation, the overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector molar ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios. Q: Will T7 DNA Ligase work in rCutSmart buffer? A: T7 DNA Ligase will work in rCutSmart Buffer, with the addition of ATP and PEG. 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.