Anza™ T4 DNA Ligase Master Mix, 200 Reactions - FAQs

T4和大肠杆菌DNA 连接酶，哪个更好用？

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。

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 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.