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WorksheetsCloning in E. coli
Total questions: 10
Worksheet time: 5mins
Which characteristic is not a typical feature of naturally occurring plasmids?
Self-replicating
Circular double-stranded DNA
Small (a few thousand bp)
Carry genes for multiple chromosomes
What is the function of the Multiple Cloning Site (MCS) in a plasmid vector?
To direct the initiation of plasmid replication
To provide selectable antibiotic resistance
To contain several unique restriction sites for easy insertion of foreign DNA
To drive the transcription of the target gene
What distinguishes an Expression Vector from a Cloning Vector?
Expression Vector has an Origin of Replication (ORI).
The Expression Vector is always non-conjugative.
The Expression Vector must contain a Promoter Region to drive transcription of the target gene.
The Expression Vector uses the lacZ gene for blue-white screening.
In the pET expression system, what molecule must be added to the medium to trigger the high-level production of the recombinant protein?
Tetracycline
RNase
Ethidium Bromide (EtBr)
IPTG
In the Alkaline Lysis Method for plasmid DNA purification, what is the fate of the linear chromosomal DNAduring the neutralization step?
It remains in solution because it is covalently closed.
It renatures correctly and remains in solution.
It precipitates along with denatured proteins and cellular debris.
It is digested by RNase A
How do researchers identify recombinant colonies (those containing the inserted foreign DNA) when using the pUC18/19 vector and the Blue-White Screening method?
They are blue because the lacZ gene is intact.
They are white because the lacZ gene is intact.
They are white because the insert causes insertional inactivation of the lacZ gene.
They are blue because the insert causes insertional inactivation of the lacZ gene.
Which of the following is a key reason for having a high copy number Origin of Replication (ori) in a cloning vector?
To tightly regulate plasmid stability.
To guarantee a high yield of the inserted DNA fragment upon purification.
To ensure the insert is in the correct orientation.
To allow the plasmid to transfer to other bacteria (conjugation).
In CsCl/Ethidium Bromide density gradient centrifugation, which component forms the lower band (highest density)?
Protein
Linear DNA
Nicked DNA
Supercoiled DNA (Covalently Closed Circular plasmid DNA)
What is the core principle used to ensure a DNA fragment is inserted into a vector in only a single, predictable orientation (Directional Cloning)?
Using a single restriction enzyme that cuts only once.
Using blunt-ended DNA fragments.
Creating both the vector and the insert with two distinct, non-complementary sticky ends.
Placing the insert within the antibiotic resistance gene.
Competent bacterial cells are those that:
Are naturally capable of conjugation.
Have undergone physical and/or chemical treatment to enhance their ability to take up DNA.
Must be treated with a high-intensity electric field only (Electroporation)
Are non-transformable and must be treated with antibiotics.
