Arthur is conducting an experi

Arthur is conducting an experiment of plasmid insertion. He hasprepared some live bacteria and gone through all essential steps ofplasmid insertion in a correct manner:

i) Cut open the plasmid by restriction enzyme and insert thegene by DNA ligase.

ii) Insert the plasmid into bacteria by heat shock.

iii) Grow the plasmid-containing bacteria in culture plate.However, after incubation, he failed to observe any successfulcloning.

Assuming the enzymes involved could function properly.

List and expain FIVE possible errors that Arthur may have andlead to this unexpected result.


Ans: 5possible errors that Arthur may have done leading to unexpectedresult are listed below

1. Arthur may have acquired Templateplasmid DNA from other researchers or may have stored it in thefreezer for months. If the purity or the concentration of plasmidDNA is compromised, cloning won’t be successful so the plasmid DNAsequence has to be verified before the start of the experiment.

2. Insert gene might have repeatedsequences leading to multiple amplicon generation during PCR. Thismight be the reason for the failure of the experiment.

3. Gene might have contained GC-richsequences. GC-rich sequences are hard to amplify during PCR andthis might have contributed to the failure of the experiment.

4. Presence of redundant restrictionsites

5. Presence of long genes. If thegene length is too large, its amplification through PCR becomesdifficult, and its insertion into the plasmid vector also becomesdifficult. This might be the reason for the failure of theexperiment.

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