- DNA LigationThis kit provides easy, highly efficient ligation.
1.Influence of Salt Concentration
We determined the influence of the DNA solution’s salt (NaCl) concentration on ligation efficiency. 5 ng of pBluescriptII 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 l 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.
2.Influence of 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. l (approx. 6.4 ng) of the reaction solution and double-stranded T Vector (50ng) were added to 3 l of Ligation high to be incubated at 16°C for each reaction time. Then, 2 l 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.