scholarly journals Optimizing DpnI Digestion Conditions to Detect Replicated DNA

BioTechniques ◽  
2002 ◽  
Vol 33 (2) ◽  
pp. 316-318 ◽  
Author(s):  
Lu Lu ◽  
Hiren Patel ◽  
John J. Bissler
Keyword(s):  
Dpni Digestion

scholarly journals Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Hui Zhang ◽  
Chang-Jun Liu ◽  
Hui Jiang ◽  
Lu Zhou ◽  
Wen-Ying Li ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
High Efficiency ◽  
Low Cost ◽  
Plasmid Vector ◽  
Efficient Tool ◽  
Overlap Extension ◽  
Cloning Strategy ◽  
Dna Fragment ◽  
Fragment Insertion ◽  
Dpni Digestion

Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.

Sign in / Sign up

Export Citation Format

Share Document