practical exam flashcards
(41 cards)
What is molecular cloning?
- a technique used to construct recombinant DNA molecules
- that can be replicated and expressed in host organisms, often E. coli.
Why are plasmid vectors commonly used in cloning?
- Plasmid vectors are small, circular DNA molecules
- can replicate independently
- contain essential features like a multiple cloning site, selectable marker, and origin of replication.
What is the purpose of PCR in molecular cloning?
- PCR amplifies the target DNA sequence
- to generate sufficient quantities for cloning into a vector.
Why is cDNA used for amplifying eukaryotic genes instead of genomic DNA?
- Eukaryotic genomic DNA contains introns,
- while cDNA is synthesized from spliced mRNA and lacks introns,
- making it suitable for expression in prokaryotes.
What are the three steps in a PCR cycle?
Denaturation, annealing, and extension.
What is the function of restriction enzymes in cloning?
They cut DNA at specific sites to generate compatible sticky or blunt ends for ligation into vectors.
Why is T4 DNA ligase used?
It covalently joins the 5’-phosphate and 3’-hydroxyl ends of DNA fragments to form recombinant plasmids.
What is the purpose of treating vectors with Antarctic phosphatase?
To remove 5’ phosphates and prevent vector self-ligation.
How are competent E. coli cells made receptive to plasmid uptake?
By chemical treatment with CaCl₂ (cold) followed by heat shock or by electroporation.
What is colony PCR used for?
To quickly screen bacterial colonies for the presence and size of an inserted DNA fragment.
How can insert orientation be confirmed in a plasmid?
By using a combination of vector- and insert-specific primers in colony PCR.
What system is used to induce recombinant protein expression in E. coli?
The T7 expression system with IPTG-inducible control.
What is SDS-PAGE used for?
To separate denatured proteins based on molecular weight.
What is the role of Coomassie Blue in SDS-PAGE?
It binds to proteins, allowing visualization after gel electrophoresis.
Why is a His-tag used in protein expression?
It allows purification by affinity chromatography and detection using anti-His antibodies.
Why is SYBR Safe used in agarose gel electrophoresis?
It stains DNA for visualization under UV or blue light and is less toxic than ethidium bromide.
What does the 6x loading dye contain and why is it used?
It contains tracking dyes and glycerol to visualize sample migration and help DNA sink into the well.
Why is Tris-acetate-EDTA (TAE) buffer used in gel electrophoresis?
It maintains a stable pH and provides ions for current flow during electrophoresis.
What is the role of DTT or β-mercaptoethanol in SDS-PAGE?
They reduce disulfide bonds, ensuring full denaturation of proteins.
Why is IPTG used in the T7 expression system?
IPTG induces expression by inactivating the lac repressor, allowing T7 RNA polymerase to transcribe the gene of interest.
What does InstantBlue Coomassie Stain do?
It stains proteins in SDS-PAGE gels for visualization without needing a separate destaining step.
Why is glycerol included in the gel loading dye?
It increases the density of the sample, helping it sink into the well.
Why is SOC medium used after E. coli transformation?
It provides rich nutrients that promote recovery and expression of antibiotic resistance before plating.
What is the purpose of a DNA ladder in electrophoresis?
It serves as a size reference to estimate the lengths of DNA fragments in a gel.
