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 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. 

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 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.

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 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.

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.