E. coli DNA Ligase
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<i>E. coli</i> DNA Ligase
Invitrogen™

E. coli DNA Ligase

E. coli DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of β-NAD between double-stranded DNAs withRead more
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Catalog number 18052019
Price (USD)
70.25
Each
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Price (USD)
70.25
Each
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E. coli DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of β-NAD between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate cohesive termini. Single-stranded nucleic acids are not substrates for this enzyme.

Applications:
Second-strand cDNA synthesis (1). T4 DNA Ligase alternative when blunt-end ligation is not required (2).

Source:
Purified from E. coli 594 (Su-) bearing λ lysogen gt4lop-11 lig+ S7 (3).

Performance and Quality Testing:
Endodeoxyribonuclease and 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested.

Unit Definition:
One unit is the amount of enzyme required to give 50% ligation of Hind III-digested λ DNA in 30 min. at 16°C in a final volume of 20 μl containing a 5´ termini concentration of 0.12 ´M (300 μg/ml).

Unit Reaction Conditions:
18.8 mM Tris-HCl (pH 8.3), 90.6 mM KCl, 4.6 mM MgCl2 , 3.8 mM DTT, 0.15 mM λ-NAD, 10 mM (NH4 )2 SO4 in 20 μl for 1 h at 16°C.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Shipping ConditionApproved for shipment on Wet or Dry Ice
EnzymeLigase
Compatible Buffer10X Reaction Buffer
Quantity100 U
Product TypeDNA Ligase
Unit SizeEach
Contents & Storage
E. coli DNA Ligase is supplied with a vial of 10X reaction buffer [188 mM Tris-HCl (pH 8.3), 906 mM KCl, 46 mM MgCl2 , 37.5 mM
DTT, 1.5 mM λ-NAD, 100 mM (NH4 )2 SO4 ]. Store at -20°C.

Frequently asked questions (FAQs)

What is the difference between T4 DNA Ligase and E.coli DNA Ligase?

The main difference between the 2 enzymes is that E. coli DNA Ligase cannot ligate blunt dsDNA fragments. Both ligases can be used to repair single stranded nicks in duplex DNA and to perform cohesive or sticky end ligations. E. coli DNA Ligase is generally used to seal nicks during second strand cDNA synthesis, since T4 DNA Ligase could result in formation of chimeric inserts.

Which T4 DNA Ligase protocol do you recommend when ligating an insert containing one cohesive (sticky) end and one blunt end?

For cloning an insert with one cohesive end and one blunt end, use the conditions for blunt ends.  The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. You should use the more stringent protocol to optimize the blunt end ligation. This usually means using more enzyme (5 U), a lower reaction temperature (14C) and a longer incubation time (16-24 hours). 

Why do I sometimes get low cDNA yield when using SuperScript Reverse Transcriptase?

Low cDNA yield can result due to several different reasons. Please see a few listed below:

(1) Poor quality mRNA: visualize total RNA on a denaturing gel to verify that the 28S and 18S bands are sharp. OD 260:280 ratio should be 1.7.

(2) Template degraded by RNase contamination: maintain aseptic conditions.

(3) Inhibitors of SuperScript II RT may be present: remove inhibitors by ethanol precipitation of the RNA preparation before the first-strand reaction. Include a 70% (v/v) ethanol wash of the RNA pellet. Test for the presence of inhibitors by mixing 1 µg control RNA and comparing yields of first-strand cDNA.

(4) RNA preparation may have coprecipitated with polysaccharides: precipitate RNA with lithium chloride to purify RNA.

(5) mRNA concentrations were overestimated: quantitate the mRNA concentrations by measuring the A260 if possible.

(6) If using 32P-isotope, it may be too old: use isotope less than 2 weeks old.

(7) Not enough enzyme was used: use 200 U SuperScript II RT/µg RNA.

(8) SuperScript II RT activity was decreased by incorrect reaction temperature: perform the first-strand reaction at a temperature between 37 degrees C and 50 degrees C.

(9) DTT was not added to first-strand reaction.

(10) TCA precipitations were performed incorrectly: adequately dry GF/C filters before immersion into scintillant.

(11) SuperScript II RT was improperly stored: store at -20 degrees C. Do not store the enzyme at -70 degrees C.

(12) The reaction volume was too large: the reaction should be done in volumes less than or equal to 50 µL.

Do both my insert and vector need to be phosphorylated for ligation?

At least one molecule in a ligase reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (phosphorylated) is most routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.

What concentration of insert and vector do you recommend for most ligation reactions?

Recommendations would vary depending on the size of the vector and insert or the nature of the insert, but for most plasmid cloning or subcloning reactions, a vector concentration of 1-10 ng/µl is recommended. Inserts should generally be 2- to 3-fold excess in molar concentration relative to the vector.