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View additional product information for T4 DNA Ligase (1 U/μL) - FAQs (15224090, 15224017, 15224025)
41 product FAQs found
两种酶的主要区别是E. coli DNA连接酶不能连接平末端dsDNA片段。两种酶均可用于修复双链DNA的单链切口以及进行粘末端连接。E. coli DNA连接酶通常被用于在第二链cDNA合成时进行切口修复，因为T4 DNA连接酶可能会导致嵌合插入的形成。
请考虑以下建议：
1.尝试不同的插入片段对载体的摩尔比。使用过量的插入片段通常会获得成功，试试1:1到15:1的插入片段：载体比例。
2.尝试增加连接反应在37摄氏度的孵育时间。
3.尝试在16摄氏度进行连接过夜（你可以在PCR仪上设置这一反应）。
确保对连接酶灭活并将连接反应产物存储在4摄氏度。
您可以设置所有下列对照或者根据面对的问题选择一个您认为最合适的对照：
1.使用环状质粒转化E.coli以评估感受态细胞的转化效率（它们获得DNA的能力如何）。
2.使用去磷酸化的载体进行转化和涂板。这将告诉你去磷酸化是否彻底以及在连接转化涂板后多大比例的克隆可能是假阳性（重新连接的载体或背景）。
3.使用T4 DNA连接酶重新连接酶切后的载体，或者lambda DNA/Hind III标记物。这将帮助你评估连接酶本身是否正常工作。
dATP是一个竞争性抑制剂。磷酸基团将降低连接效率。连接缓冲液中的去垢剂可能不会影响酶活性。高浓度(0.2 M)的Na2+, K+, Cs+, Li+, 和NH4+几乎可以完全抑制酶活性。多胺、精胺和亚精胺也是抑制剂。
在一个连接反应中，至少有一个分子（例如插入片段或载体）必须是磷酸化的。连接反应取决于DNA分子上5’磷酸的存在。去磷酸化载体与限制性内切酶消化生成的插入片段（磷酸化的）结合，是最常见的方式。尽管DNA只有一条链可连接在接合点，但是也可形成稳定环状结构的分子，前提条件是插入片段足够长，双链杂交产生的力量能够维持分子的环状结构。
推荐浓度取决于载体和插入片段的大小或插入片段的性质，但对于大多数质粒克隆或亚克隆反应来说，推荐的载体浓度为1-10 ng/µl。插入片段的摩尔浓度通常超过载体的2倍或3倍。
这取决于您的应用。对于含有黏性末端的双链DNA片段之间的连接，两种酶都能使用。大肠杆菌DNA连接酶需要β-NAD存在，而T4 DNA连接酶需要ATP。然而，只有T4 DNA连接酶可以连接平末端DNA片段；大肠杆菌连接酶不可用于这类片段的连接。
大肠杆菌DNA连接酶通常用于第二链cDNA合成中修复缺口。在第二链cDNA合成中，T4 DNA连接酶不可代替大肠杆菌DNA连接酶，因为T4 DNA连接酶对平末端ds cDNA片段连接的功能，可能导致形成嵌合插入。
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.
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).
Make sure you have inactivated the ligase and store the ligation reaction at 4 degrees C.
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.
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.
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.
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).
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.
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.
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.
To test for the presence of ligation inhibitors, perform a ligation reaction in which some of the vector or insert DNA is included along with some marker DNA such as lambda DNA/Hind III Fragments. If ligation of the DNA marker fragments occurs alone but is not observed when other DNA is added, then a diffusible inhibitor is present in the vector or insert DNA.
To purify and remove inhibitors, extract the DNA with buffer-saturated phenol, then extract with chloroform:isoamyl alcohol, and precipitate with ammonium acetate and ethanol. Be sure that the DNA is free of phenol and that the phosphate concentration is less than 25 mM and the NaCl concentration is less than 50 mM. Also, be sure that the DNA is free of contaminating DNA that might compete for ligation to the insert or vector (e.g., linker fragments, DNA fragments from which the insert was completely purified).
If restriction endonucleases are present, causing redigestion of ligated products, your ligation will also be inhibited. After digestion of the vector and insert DNA, remove restriction endonucleases by extraction with buffer-saturated phenol, extraction with chloroform:isoamyl alcohol, and ethanol precipitation.
Ligation reagents may be tested by performing a ligation reaction with a molecular size marker such as the 1 Kb DNA Ladder or lambda DNA/Hind III Fragments. Compare the ligation reaction products to unligatedDNA on an agarose gel. The ligation reaction should contain a high molecular weight smear and few low-molecular weight bands. If the marker ligation does not work, use fresh ligase.
Another reason for low activity could be degradation of the ATP in the reaction buffer; use 5X ligase buffer that is less than 24 months old. The buffer should be stored at -20 degrees C.
Several ligation controls may be necessary to identify the source of ligation problems.
Recommendations for problems with no colonies after transformation:
1. Test the transformation efficiency of the competent cells using a supercoiled vector, or the control DNA provided with Invitrogen competent cells. Perform a transformation reaction and plate the number of cells that is expected to generate 50-100 colonies per plate, based on the anticipated transformation efficiency of the competent cells. The expected number of colonies should be seen, indicating that the competent cells are transforming with high efficiency. The control DNA provided with Invitrogen competent cells is supercoiled monomer; vector DNA preparations that contain other forms will not transform as efficiently. Transformation efficiencies will be up to 10-fold lower for ligated vectors than for intact control DNA.
2. Restriction endonuclease-digested, re-ligated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Re-ligation of vectors with cohesive ends should result in less than or equal to 50% of the number of colonies obtained with supercoiled vector DNA, indicating that the components of the ligation reaction are working; re-ligation of vectors with blunt ends should yield 10% to 20% of the number of colonies obtained with supercoiled vector DNA. This is an appropriate control only with vectors that have been digested with a single restriction endonuclease; double-digested vectors may not re-ligate because the ends are incompatible and the small DNA fragment that is released from between the two sites is sometimes lost during ethanol precipitation of the DNA.
For observation of a high number of background colonies:
1. Restriction endonuclease-digested vector. Perform a transformation with an amount of restriction enzyme-digested vector DNA equivalent to that contained in the fraction of the ligation reaction being used for the experimental transformations. Few or no colonies should be seen, indicating complete restriction endonuclease digestion of the vector. The presence of colonies indicates incomplete digestion of the vector that will cause a background of colonies containing non-recombinant vector in the experimental transformations.
2. Restriction endonuclease-digested, dephosphorylated, re-ligated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Few or no colonies should be observed, indicating complete dephosphorylation of the vector - a dephosphorylated vector should not be re-ligated by T4 DNA ligase.
3. No DNA transformation control. Perform a mock transformation of competent cells, to which no DNA is added. No colonies should be seen, indicating that the selection antibiotic on the agar plates is potent and that the competent cells are pure.
Ligation reactions are best analyzed by actual transformation of bacteria, since not all of the high molecular weight forms created in a reaction (seen in gel analysis) will transform cells efficiently.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The PTH column is worn out; you should replace it.
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.
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.
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.
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.
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.