T4 DNA Ligase (5 U/μL)
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T4 DNA Ligase (5 U/μL)
Invitrogen™

T4 DNA Ligase (5 U/μL)

T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxylRead more
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Catalog number 15224041
Price (USD)
214.00
Each
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Price (USD)
214.00
Each
Add to cart
T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes (1). Single-stranded nucleic acids are not substrates for this enzyme. A T4 DNA Ligase Technical Bulletin is available.

Applications: Cloning (blunt-end or cohesive-end ligation) (2). Adding linkers or adapters to blunt-ended DNA (2).

Source: Purified from E. coli œ lysogen NM989.

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

Unit Definition: One unit catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into ATP in 20 min. at 37°C. (One unit is equal to approximately 300 cohesive-end ligation units.)

Unit Reaction Conditions: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2 , 10 mM DTT, 66 μM ATP, 3.3 μM 32 P-labeled pyrophosphate, and enzyme in 0.1 ml for 20 min. at 37°C.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Concentration5 U/μL
Shipping ConditionDry Ice
EnzymeLigase
Compatible Buffer5X Reaction Buffer
Quantity250 U
Product TypeT4 DNA Ligase
Unit SizeEach
Contents & Storage
T4 DNA Ligase is supplied with a vial of 5X reaction buffer [250 mM Tris-HCl (pH 7.6), 50 mM MgCl2 , 5 mM ATP, 5 mM DTT, 25% (w/v) polyethylene glycol-8000]. 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.

How can I optimize my ligation reaction?

Please consider the following suggestions:
1– Try different molar ratios of insert to vector. Having an excess of insert is usually what will work, try 1:1 to 15:1 insert:vector.
2– Try increasing the time of the ligation at 37 degrees C.
3– Try performing the ligation at 16 degrees C overnight (you can set it up on your PCR machine).

I cannot transform my cells right away. Can I store my ligation reaction? If so, at what temperature should I store it?

Make sure you have inactivated the ligase and store the ligation reaction at 4 degrees C.

What kind of controls should I have for restriction cloning?

You can have all of the below controls or select the one you consider the most appropriate to the problem you are facing:
1– Transform the E. coli with circular plasmid to assess the competency of the cells (how well they are taking up DNA).
2– Transform and plate the dephosphorylated vector. It will help you assess how well the dephosphorylation worked and what proportion of colonies in your ligation transformation plate could be false positives (re-ligated vector or background).
3– Use T4 DNA igase to re-ligate your cut vector, or lambda DNA/Hind III marker. It will help you assess whether the ligase itself is working properly.

What are common inhibitors of the T4 DNA ligase?

dATP is a competitive inhibitor. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2M) Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.

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