제품 설명TOPspeed™ DNA Ligation Kit는 cohesive end를 지닌 DNA 단편의 실온 상에서 5분 이내의 신속한 ligation을 위해 조제되었습니다. 이 제품은 T4 DNA Ligase (Cat. # M001) 및 2X TOPspeed ™ DNA Ligation buffer를 사용합니다.
제품 특징
- 5-min ligation reaction with all types of DNA fragment
- Reaction at room temperature
- Rapid and efficient TOPspeed™ Ligation buffer
제품 응용
- General cloning
- Blunt-end cloning
- TA cloning
- Library construction
- Linker ligation
주의 사항
-사용 전 2X TOPspeed ™ DNA ligation buffer를 실온으로 예열해 주십시오. 반응 당 20μl 이상을 사용하는 것이 좋습니다. 완충액의 반복적인 동결-해동과정을 피하십시오.
-Ligation 전에 vector와 DNA insert의 mole number를 DNA의 농도 측정을 바탕으로 계산하십시오. Vector DNA보다 2 ~ 3 배 (sticky end DNA ligation) 또는 5 ~ 6 배 (blunt end ligation) 더 많은 삽입 DNA를 사용하십시오.
-TOPspeed ™ DNA ligation mixture을 가열하면 Transformation 효율이 급격히 떨어집니다.
-1 시간 이상 반응하면 transformation 효율이 떨어질 수 있습니다. 따라서 5-30 분의ligation 반응 후에 Transformation을 수행하는 것이 좋습니다.
Figure 1. Ligation of blunt-end fragments using TOPspeed™ DNA Ligation Kit.
Bacteriophage λ DNA was digested with EcoR V (Cat.# R018) to generate blunt-ended restriction fragments. EcoR V-cleaved λ DNA (500 ng) were ligated by the use of TOPspeed™ DNA Ligation Kit (Cat.# EZ003). Periods of incubation are as indicated (in min). Ligation products were analyzed in a 1% agarose gel. Lane 1, EcoR V-cleaved λ DNA only as a negative control; Mw, 1 kb (+) Ladder Marker (Cat.# DM003).
Figure 2. Enhanced ligation efficiency with TOPspeed™ DNA Ligation kit.
Vector DNAs were cleaved with either Hind III (Cat.# R008), EcoR V (Cat.# R018), or Hind III/Xba I (Cat.# R013). Vectors digested with Hind III or EcoR V were dephosphorylated prior to ligation reaction. The vector DNAs prepared this way were mixed with insert DNAs precleaved with the corresponding restriction enzyme(s). Ligation reactions were carried out with T4 DNA Ligase (Cat.# M001) at 16℃ for 16 hr (green box, standard) or with TOPspeed™ Ligation Kit (Cat.# EZ003) at 25℃ for 5 min (red box, TOPspeed). Ligation products were directly transformed into chemically competent E. coli DH5 (Cat.# CP010) to measure ligation efficiency. Fold increases in the numbers of transformants were presented; the number of transformants obtained from standard ligation procedure was assumed to be 1.
