Review on Restriction Digestion

Running head: REVIEW ON RESTRICTION DIGESTION
REVIEW ON RESTRICTION DIGESTION
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REVIEW ON RESTRICTION DIGESTION
1. Partial digestion of vector DNA pUC18 with HindIII involve an incomplete digestion of
the plasmid DNA vector pUC18 that will result in cut of DNA vector at multiple site that
may or may not involve the desired location (De Paoli, Tuskan & Yang, 2016). pUC 18
vector has one restriction site for HindIII site where bacteriophage lamda DNA has to be
ligated. In order to identify this Agarose Gel Electrophoresis should be performed. After
the digestion process, the fragmented DNA is run on the gel, and then auto radiographing
is performed. The result will show more number of bands than the required one. The size
of fragment can be confirmed by using DNA marker of known size (Li et al., 2016). Due
to partial digestion bacteriophage will not be ligated at the desired site, resulting improper
insert.
2. Ampicillin is known to bind and inhibit the enzymes that are present in the bacterial cell
membrane and is involved in production of cell wall of bacteria. The gene codes for an
enzyme that hydrolyse the B-lactam ring of ampicillin (Santajit & Indrawattana, 2016).
The competent cell having ampicillin resistance gene can be made confirm by growing
the cell in ampicillin containing media. If the cell is having the ampicillin resistance gene
it will block the action of the antibiotic and allow production of enzyme that will make
bacteria cell wall. Thus, cell growing in the ampicillin media will confirm that they
contain ampicillin resistant contaminants.
3. Absence of the Tranformant can be the indication that ligation of insert gene has not
taken place at the desired location. This is the problem as because, this confirms that
whole digestion process has gone wrong. The problem can be either in transformation or
in ligation process. If there is incomplete digestion, no growth of colonies is observed.
However, if there is problem in transformation step, there is very high number of colonies