Question

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

i) Cut open the plasmid by restriction enzyme and insert the gene 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 successful cloning.

Assuming the enzymes involved could function properly.

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

Answer 1

Ans: 5 possible errors that Arthur may have done leading to unexpected result are listed below-

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

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

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

4. Presence of redundant restriction sites

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