Ligation high is a ligation kit consisting mainly of T4 DNA Ligase. Composition has been optimized to improve stability after freezing/thawing cycles and enhance reaction efficiency. All of the reaction buffers, rATP, and DTT, etc. required for ligation are supplied with the kit. Ligation high provides better results than T4 DNA Ligase alone, just by adding to a DNA sample solution from a half up to an equal volume. Repeated freezing/thawing cycles may affect the reagent added in some commercially available kits to enhance ligation efficiency, degrading it and actually reducing efficiency. This kit, however, does not exhibit such a tendency and allows highly effective ligation at all times.
DNA LigationThis kit provides easy, highly efficient ligation.
We determined the influence of the DNA solution’s salt (NaCl) concentration on ligation efficiency. 5 ng of pBluescript®II digested with SacI was suspended in 5 ml of TE Buffers different in NaCl concentration.
Half and equal amounts of Ligation high were added to react at 16°C for 30 minutes and then 2 µl was used for transformation to determine the number of colonies obtained. The result showed ligation efficiency decreased as NaCl concentration increased.
When using DNA obtained in a reaction mix with high salt concentration such as restriction endonuclease Buffer H for ligation, we recommend substitution of the solution with TE Buffer, etc.
Reaction Time on PCR Products in TA Cloning
PCR products were ligated into T vector using this kit. PCR was performed with 500 bp of λ DNA as the target using Taq DNA Polymerase. µl (approx. 6.4 ng) of the reaction solution and double-stranded T Vector (50ng) were added to 3 µl of Ligation high to be incubated at 16°C for each reaction time. Then, 2 µl of the reaction solution was used for transformation to determine the number of white and blue colonies obtained. As a result, ligation efficiency increased as reaction time increased reaching its peak at 16 hours. Moreover, no difference was observed in the development of blue colonies and white colonies when the reaction time was increased to 2 hours or longer. To obtain a high insertion rate using Ligation high for TA Cloning of PCR products, we recommend reaction times longer than 2 hours.
3.Examination of Reaction Time
Ligation was performed with λ/Hind III at 16°C for each reaction time and electrophoresis performed after EtOH precipitation.
The picture shows that Ligation high obtained ligation in 5 minutes.
Feature and Advantages:
Easy to Use
Reactions using this kit perform ligation just by mixing Ligation high with a sample solution.
Provides 50-fold or higher efficiency compared to use of T4 DNA Ligase alone.
Ligation activity is not lost even after 50 freezing/thawing cycles
In general add an equal amount of Ligation high to 15 µl of a DNA sample. To clone a sample digested with a restriction endonuclease into a vector, the high purity of the DNA sample may provide sufficient results even when using a halved amount of Ligation high. To perform TA cloning or when using a sample obtained from agarose gels, we recommend Ligation high be added in equal volume.
When using a sample for electroporation after a ligation reaction, the sample should be desalinated by ethanol precipitation or using a kit, etc. Similarly, for the calcium chloride method, when the DNA sample used in ligation is dissolved in a buffer with a potentially high salt concentration, it is advisable to perform desalination.
Temperature and Reaction Time
Generally, better results will be obtained from reactions performed at 16°C for 30-60 minutes. For TA cloning, it is better to perform reactions for 2 hours or more. If low ligation efficiency is obtained, increasing the reaction time may produce better results. Efficiency should be unaffected in reactions of several hours even at room temperature.
|Ligation high||TYB-LGK-101||50 RXN||€277|
|Ligation high Ver.2||TYB-LGK-201||750 UL||€294|