[Bioengineering]Assume the digestion of isolated plasmid did not produce the ...
Assume the digestion of isolated plasmid did not produce the expected band pattern. (After preparation of plasmids by alkaline lysis and column purification, followed by digestion of plasmid DNA by restriction endonuclease and gel electrophoresis)a) Explain two potential causes.b) How would each of these causes be reflected in the sequencing results?static/image/hrline/line3.png
A. Reasons for not getting desired bands after plasmid digestion:1. Incomplete or no digestion a) Inactive restriction enzyme b) Suboptimal reaction conditions c) Enzyme activity blocked by DNA methylationd) Substrate DNA structuree) Insufficient incubation time f) Low enzyme concentration g) Contaminated template 2. Unexpected cleavage patterna) Gel-shift Effectb) Star activity and other related causesc) Contamination and unexpected recognition sitesB. Suppose the undesired bands appeared due to contaminated template/ DNA. If we add a primer designed for binding at a specific region of the template for sequencing, then the primer will not bind at that specific site and non- specific binding at other places will result into incorrect sequencing result.Apart from that, due to use of inactive restriction enzyme or due to suboptimal reaction conditions, the sequencing results may get hampered. Suppose, the plasmid was not cleaved completely due to inefficient cleavage activity for any of the above reasons, then the digested (may be double digested) plasmid may get self re-ligated and due to which the designed primer for sequencing will not bind or amplify anymore. Thus we may get wrong sequencing result by doing the sequencing, as now the primer may amplify a wrong part of the plasmid/ DNA.Explanation:Approach to solving the question: Restriction Digestion, Genetic engineering, Cloning of plasmid and methods Detailed explanation: Already explained above. You need to be thorough about the cloning procedure and troubleshooting for the wrong result. Possible explanation along with answer has been described above briefly.
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