Q1: What are some potential problems with the ligation reaction using T4 DNA Ligase that can

lead to transformation failure? A1: * 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 g/ml. * The ligated end was a single base overhang. Use up to 5 l concentrated ligase at 16C 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 l to 50 l competent cells. * The insert was large and the conditions didn't allow circularization. Reduce the insert concentration and use concentrated ligase at 16C 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. Q2: What are some other problems that should be considered when trouble shooting a transformation problem? A2: * 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. Q3: What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation to fail? A3: * 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. Q4: What controls should be run to test the cells and DNA when using T4 DNA Ligase? A4: 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. Q5: When should T4 DNA Ligase be the enzyme of choice? A5: 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. Q6: Can the T4 DNA Ligase be used with the Quick Ligase buffer? A6: Yes, concentrated T4 DNA Ligase can be substituted directly. When using regular T4DNA 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. Q7: What is the definition of a Weiss Unit and a Cohesive End Unit? A7: 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 37C, 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 M (300 g/ml)) in 20 l of 1X T4 DNA Ligase Buffer in 30 minutes at 16C. Q8: What is the difference between the two definitions and why does NEB use the Cohesive End Unit? A8: 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. Q9: How much DNA should be used in a ligation using T4 DNA Ligase? A9: The unit definition uses 0.12 M (300 g/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 g/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. Q10: Can T4 DNA Ligase be used in other NEBuffers? A10: Ligation can also be performed in any of the four standard restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP. Be sure to use riboATP (NEB# P0756) as deoxyriboATP will not work. When using NEBuffer 3 high levels of salt in the DNA preparation may decrease ligation efficiency. Q11: Can T4 DNA Ligase be heat inactivated? A11: 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.