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Invitrogen™ Ambion™ T4 RNA Ligase, cloned, 5 U/μL

Catalog No. AM2141
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2,500 units, 5 μL
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AM2141 2,500 units, 5 μL
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Catalog No. AM2141 Supplier Invitrogen™ Supplier No. AM2141
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Description

Catalyzes formation of a phosphodiester linkage between a 5'-phosphoryl-terminated nucleic acid donor and a 3'-hydroxyl-terminated nucleic acid acceptor

  • Substrates include RNA, DNA, and oligonucleotides. One tube containing 500U (5U/μL) is provided along with 10X Reaction Buffer
  • The enzyme can be used for tagging the 5′ ends of mRNA with oligonucleotides for mapping studies (RACE), 3′-end labeling RNA molecules, and circularizing RNA and DNA molecules
  • Unit Definition: One unit catalyzes the formation of 1nmol of [5′-32P]-rA12-18 into a phosphatase-resistant form in 30 minutes at 37°C

DNA & RNA Purification & Analysis, Nucleic Acid Labeling & Oligo Synthesis, Oligo Labeling, RNA Labeling

Specifications

Concentration 5 U/μL
Content And Storage T4 RNA Ligase and 10X Reaction Buffer should be stored at –20°C.
Format Tube
Enzyme Ligase
Fidelity (vs. Taq) 10 X
No. of Reactions 250
Polymerase SP6 RNA Polymerase
Product Line Ambion
Product Type T4 Rna Ligase
Quantity 2,500 units, 5 μL
Shipping Condition Dry Ice
Source RNA
Form Solution
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Frequently Asked Questions (FAQs)

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.

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