Article History

Received 6 May 2020
Received in revised form 15 May 2020
Accepted 10 June 2020
Available online 30 June 2020

Keywords

Multicopy clones, Post Transformational Vector Amplification, recombinant puc57 vector, Real-Time PCR, DH5a strain, Ct value.

Determination of multicopy clones containing recombinant papain gene by Real-Time PCR

Esha Rajput1*, Neha Agarwal2 and Sachin Chauhan3

1Department of Biotechnology, Hindu college, Moradabad.

2Department of Biotechnology, Hindu College, Moradabad.

3Taq Gene Training and Research Institute (TGTRI), Dehradun.

*Corresponding author :rajputesha999@gmail.com

ABSTRACT: Multi copy clones also known as “jackpot clones” are transformed cells with a recombinant gene having greater number of recombinant gene copy as compare to normal one. Post Transformational vector amplification (PTVA) is a technique for production of multicopy clones. In PTVA the selection of transformed cell is done on agar medium containing the increasing concentration of antibiotic.  A recombinant PUC57 vector containing the artificial synthesized papain gene was used to transform the DH5 strain of E.coli. The Transformed cells were selected on LB agar plates with increasing concentration of ampicillin antibiotic (100g/ml, 250g/ml, and 500g/ml), the number of colonies were reduced as ampicillin concentration was increased. Plasmid DNA isolation was carried out from Qiagen-Qiaprep-mini DNA isolation kit. Real time PCR is performed to identify multicopy clone on each plates. The average cycle threshold value i.e. Ct value for 500g/ml LB-Amp Plate is 19.46, for 250g/ml LB-Amp Plate is 22.40, and 100g/ml LB-Amp Plate is 24.68. The Real-time PCR results indicated the higher level of gene expression in cells grown in higher antibiotic concentration.

 

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