Protocol.html

PROToCOLS

1.     Protocol for Cloning Experiments

·        Protocol for PCR Amplification, Competent Cell Preparation, and Transformation was followed as given in Molecular Cloning: A Laboratory Manual, 3^rd edition Sam brook.

·        All Restriction Endonuclease used are of Thermo Fisher Scientific and T4DNA ligation Enzyme is of New England Bio labs and all reactions are set as per guidelines of the supplier.

 

2.  Promoter Characterization

We had done in In-Silico Characterization of Promoter by validating the cis-element of the promoters by using Plant Care.

(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)

Functional validation of characterized MMV promoter via protoplast assay in tobacco had done by expertise of the plant biotechnology lab, Department of Botany D.E.I, Dayalbagh, Agra, Protocol had not been shared due to publication and patenting issues.

3.  Protein Characterization

·        Quantity Estimation of Protein: For quantify the expressed protein Bradford Method is used as given on the following link -(http://www.bio-protocol.org/e45).

·        Quality Estimation of protein:  The quality of expressed protein was done by SDS-PAGE.

·        Protocol for the SDS-PAGE is followed as given on the following web-link- (http://www.assay-protocol.com/molecular-biology/electrophoresis/denaturing-page)

 

4.  Immobilization of Cells Expressing our Protein of Interest

 

Cells of E. Coli Bl-21 expressing our desired protein are immobilized using the protocol given by Dhall et al., 2012.

(http://sci-hub.cc/http://pubs.rsc.org/en/content/articlelanding/2012/ay/c2ay25960e/unauth#!divAbstract

 

5.  Efficacy Testing of Beads by treating with water samples possessing heavy metals contamination

Concentration of heavy metals in samples treated with our fabricated bio-beads (BECO) are analyzed by ICP-OES  of Agilent Technology.

 

References:

1.       Dhall P, Kumar R and Kumar A. 2012. BOD beads – a ready to use seeding material for estimation of organic load of wastewater. Anal. Methods, 2012, 4, 4101. DOI: 10.1039/c2ay25960e

2.       He, F. (2011). Bradford Protein Assay. Bio-protocol Bio101: e45. DOI: 10.21769/BioProtoc.45.