Sigma-Aldrich, USA
Quick LinkTM DNA ligation Kit
LIG2
This assay kit was used for joining DNA fragments into a cloning vector.
Cloning (joining blunt or cohesive-end fragments of DNA into a cloning vector)
Restriction enzyme (EcoRI and Not1) digested DNA fragments and cloning vector (pCMv6).
Quick LinkTM DNA Ligation Kit was used for obtaining maximum ligation and transformation yield. DNA fragment was obtained from DNA and digested by two restriction ezymes. These DNA fragments were separated in low melting agarose gel and and selected and purified and dissolved in TAE buffer, pH 7.4-7.6. Ligation Buffer B was added and ligation was performed according to the manufacturer’s instructions.
None
Ligation product was transformed in competent E. coli (DH5a), selected for positive colonies and amplified. Plasmid DNA was E. coli which was purified with mini prep kit and resolved in 1 % agarose gel electrophoresis. Ligation was also confirmed by PCR using specific primers for insert.
Self annealing was not observed
Optimization of protocol takes time.
Quick LinkTM DNA ligation Kit is good for efficient ligation