Cloning & Transgenesis
Open Access
ISSN: 2168-9849
ISSN: 2168-9849
Syed Naqui Kazim
India
Research Article
Designing and Reconstruction of pcDNA 3.0 Mammalian Expression Vector with its Multiple Cloning Sites by Directional Cloning Method
Author(s): Sheeba Naaz and Syed Naqui KazimSheeba Naaz and Syed Naqui Kazim
Traditional cloning is the cloning in which we use restriction endonucleases to produce DNA fragments with specific complementary end sequences that can be joined together with a DNA ligase enzyme, prior to the transformation. The purpose of this study was to reconstruct the mammalian expression vector by replacing the junk DNA with its natural multiple cloning sites. Plasmid was propagated and digested with Xho1 and Kpn1 enzymes from both sides of junk DNA a 600 bp DNA was cut from the vector and then ligation of 30 bp primer which was designed similar as its multiple sequence sites. Correct insertion was confirmed by plasmid isolation, plasmid colony PCR, PCR of cloned plasmid and comparison on gel electrophoresis with the original one and finally by sequencing. An extra DNA fragment of 600 bp was cut from the vector after restriction digestion. Ligated colonies appeared on the agar.. View More»
DOI:
10.4172/2168-9849.1000162