Invitrogen™ T4 DNA Ligase Buffer

Catalog No.	Quantity
46-300-018	2 x 1 mL

Catalog No. 46-300-018 Supplier Invitrogen™ Supplier No. 46300018

Description

T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes

• As supplied with T4 DNA Ligase
• Formulation: 250mM Tris-HCl (pH 7.6), 50mM MgCl₂, 5mM ATP, 5mM DTT, 25% (w/v) polyethylene glycol-8000

Cloning (blunt-end or cohesive-end ligation), Adding linkers or adapters to blunt-ended DNA, ChIP-on-Chip, Chromatin Biology, Cloning, RNAi, Epigenetics & Noncoding RNA Research, Restriction Enzyme Cloning

Order Info

Shipping Conditions: Approved for shipment on wet or dry ice

Specifications

• Content And 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.
• Shipping Condition: Approved for shipment on Wet or Dry Ice
• Compatible Buffer: 5X Reaction Buffer
• Quantity: 2 x 1 mL
• Product Type: T4 DNA Ligase Buffer

Frequently Asked Questions (FAQs)

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.

Do both my insert and vector have to be phosphorylated for successful 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.

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 is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

What are the recommended conditions for blunt-ended ligations?

Generally, ligations are done in a 20 µL volume. Use a total of 100 to 1000 ng of DNA with an insert to vector ratio of 3:1. Add 1.0 units (Weiss) ligase to the reaction. Incubate at room temperature for 4 h or overnight at 14-16 degrees C.

Ideally, assemble several reactions with varying ratios of vector:insert (i.e. 3:1, 5:1, 10:1, 20:1, etc.) to determine the optimal ratio for ligation.

Thermo Fisher Scientific offers T4 DNA ligase at two concentrations: 1 U/µL (Cat. No. 15224-017) and 5 U/µL (Cat. No. 15224-041). When performing blunt or TA cloning ligations, the higher concentration of ligase is generally preferred since ligating a blunt or single base overhang requires more enzyme.

What are the recommended conditions for cohesive-end ligations?

Generally, ligations are done in a 20 µL volume. Use a total of 10 to 100 ng of DNA per reaction with an insert to vector ratio of 3:1. Add 0.1 units (Weiss) ligase to the reaction. Incubate at room temperature for 30-60 minutes.

Optimal ligation may occur at other ratios (e.g. 1:5, 1:10). If possible, assemble several ligation reactions of varying insert to vector ratios in order to reveal the optimal ligation conditions.

Thermo Fisher Scientific offers T4 DNA ligase at two concentrations: 1 U/µL (Cat. No. 15224-017) and 5 U/µL (Cat. No. 15224-041). When performing blunt or TA cloning ligations, the higher concentration of ligase is generally preferred since ligating a blunt or single base overhang requires more enzyme.

Which is better to use, T4 or E. coli DNA ligase?

It depends on your application. For ligation of dsDNA fragments with cohesive ends, either enzyme can be used. E. coli DNA ligase requires the presence of beta-NAD, while T4 DNA ligase requires ATP. However, only T4 DNA ligase can join blunt-ended DNA fragments - E. coli ligase is unable to join such fragments.

E. coli DNA ligase is generally used to eliminate nicks during second-strand cDNA synthesis. T4 DNA ligase should not be substituted for E. coli DNA ligase in second-strand synthesis because of its capability for blunt end ligation of the ds cDNA fragments, which could result in formation of chimeric inserts.

Why is ATP present in the reaction buffer for T4 DNA Ligase?

ATP is necessary for enzymatic function. It is involved in phosphorylating the ligase prior to the ligation reaction. Ligation efficiency is markedly reduced by removing ATP from the reaction. It is important, therefore, to handle the buffer appropriately in order to minimize degradation of ATP.

What causes synthesized \peptides to be over or under the expected weight when post-cleavage mass spectral analysis is performed? What causes multiple peaks for one peptide, by RP-HPLC?

Overweight: Most often reflects failure to remove all the side-chain protecting groups from all amino acids ("incomplete cleavage"). If insufficient amounts of scavengers were used, these protecting groups may reattach, OR permanently attach to another amino acid. If this occurs, it may be necessary to modify the system used for cleavage. Increased time, better mixing, or increased scavengers may be needed. This may be best determined by trial and error.
Underweight: Usually reflects a problem during the synthesis. If the run was done with conductivity or UV monitoring during Fmoc removal, it may be possible to modify the subsequent synthesis to avoid problems at specific areas of the sequence. An analysis of the probable secondary structure of the peptide chain may also be helpful for future strategies.

How can peptide synthesis amino acid cartridges leak and spill solvents during their activation and transfer to the RV?

If these cartridges are being reused, the NMP can cause them to swell, and they no longer fit or slide well in the guideway. If the guideway or the exterior of the needles have became dirty, this can also lead to misalignment. And if you forgot to remove the metal cap, the needle cannot penetrate the septum - this may cause a spill OR stop the run.

How often should the sound of the vacuum assist be heard on the 433A?

At most, once or twice a day. If more frequent, there may be a gas leak. Nitrogen pressure is used to generate the vacuum, which assists the opening of the valves. If there is no apparent gas leak, then it is possible that a valve has failed and the solvent leakage has damaged the vacuum ballast. Both the vacuum system and the valve block need inspection.

Why doesn't the 433A manual or the "quick start card" mention the need for an extra AA cartridge in the guideway at the start of a sequence?

The newest 433A User's Manual does cover this. The barcode reader is one position ahead (left) of the needle position. The extra (empty) is needed to prevent advancement of the first cartridge until after it is read.

Why is the conductivity so high in my peptide synthesis (monitored during deprotection)?

The meter detects any ionic species. A common cause of higher than expected values is a leak of a small amount of resin from the RV into the lines and up to the in-line filters. The use of old or poor quality piperidine or NMP may also give a high background. Standard conductivity measured in micro Siemens/cm is much higher than the sensitivity of this cell. A very small amount of ionic material caused a large change in the reading. Occasionally, Fmoc amino acids have ionic contaminants which give high readings. In-line filters may also be contaminated.

What can be done when I observe errors on the 433A such as "chemistry not declared" (CND), a "PAUSE" that will not unPAUSE, a "run cannot be sent", or during a send the chirps are slower than usual?

The problem occurs when a 433A connected to a Macintosh computer interprets a signal from the computer as a "reset". Some "fixes" have been successful, particularly installation of a special optical isolator on the 433A. When the instrument has frozen in a run, the run needs to be stopped and re-started, often with a "COLD REBOOT". To prevent the problem, it is strongly suggested that no network connection be attached to the computer during a run. No other software should be run, or even loaded to the hard drive of the computer.

How can I tell whether or not detergent in a sample can be removed with Prosorb Sample Preparation Cartridges?

If the detergent identity is known, look up its CMC (critical micellar concentration); a table of these is listed in the Biological Detergents section of the Sigma-Aldrich catalog. If you are above the CMC, the detergent forms micelles that cannot be removed by the filtering membrane, so the sample must be diluted prior to application to the Prosorb Sample Preparation Cartridges.

How can I tell whether or not a reagent bottle is pressurizing correctly when using the Procise System?

You can backflush the bottle's pickup line in manual control (there is a specific function for each bottle position on the Procise System) and observe its bubbling, which should slow and then stop within a short period (depending upon how full the bottle is) as it pressurizes with argon. If it continues to bubble, either the cap assembly is leaking or the pressurizing or venting valves for the bottle are.

I am using the Procise System and have increased the final %B in the gradient to separate LYS from LEU, but now all the late PTH amino acid peaks are crushed together. What can I do?

The PTH column is worn out; you should replace it.

How can I improve peak resolution between ASP and DTT or ASN when using the Procise System?

You can move ASP (and GLU) away from DTT and to later retention times by reducing slightly the pH of Solvent A3. This is best accomplished by adding a small amount of TFA (R3)--about 50 to 100 ul/liter of Solvent A3. In the Procise System cLC, when ASP and ASN are too close together, the problem can usually be resolved by replacing the guard column (part no. 401883). Decreasing initial %B in the gradient (e.g., from 10% to 8%) will move both DTT and ASP to later retention times.

What can cause broad dips or cyclical peaks in the baseline when using the Procise System?

This can be due to pump irregularities, often caused by worn seals. A more or less regular "sawtooth" pattern is usually indicative of a failed dynamic mixer.

I want to make peptide amides, but your amide resin has no amino acid attached. Why not? Do I need to do anything special?

There is no amino acid attached because one is not needed. The amide linker has a free amine which is protected by an Fmoc group. Upon removal of the Fmoc group, an amide bond may be formed with the incoming activated amino acid. Nothing special needs to be done, although you must tell your synthesizer you are using an amide support and/or the first amino acid is not on the support. Standard cleavage protocols may be used.

How many degenerate sites are allowed in a 25-mer present at a total concentration of 1 micromolar (100 pmoles/100 mL)?

Primer mixtures with 256-fold and 32-fold degeneracies have been used [see Mack DH, Sninsky JJ (1988) Proc Natl Acad Sci USA 85:6977-6981 and Lee et al. (1988) Science 239:1288-1291.] We recommend that users synthesize pools of no more than 32- to 64-fold degenerate primers, making additional pools separately to account for all possible degeneracy. A matrix should be set up so that degeneracy is no more than 2-fold at each site, with all sites in the matrix run at the same time. Inosine may also be used for the degenerate positions in the primer. Performing touchdown PCR may help increase the specificity of degenerate primer PCR amplification.

What are some of the problems associated with sticky-end cloning?

The amplified DNA needs to be purified from the PCR mixture components prior to cloning. The dNTPs carried over from the PCR are competitive inhibitors for ATP in the ligation reaction.

If during synthesis of the PCR primers their chemical integrity has been compromised by either a base substitution or modification, the enzyme recognition site may in actuality not exist. If this is the case, PCR products will be resistant to digestion with restriction enzymes. It may be necessary to use a higher concentration of the restriction enzyme and to incubate at the appropriate temperature overnight to ensure cutting.

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.