bio practicum plz pass Flashcards
(22 cards)
❓ What is the purpose of molecular cloning?
To create multiple identical copies of a specific DNA sequence (gene) by inserting it into a plasmid vector and propagating it in a host organism (usually bacteria).
🧬 In the context of molecular cloning, what is a plasmid?
A small, circular piece of DNA that replicates independently of chromosomal DNA in bacteria. It is used as a vector to carry foreign DNA into a host cell.
Define these features of a plasmid: Selectable marker, origin of replication(ori), multi-clone site(mcs)/poly-cloning site
- Selectable marker: A gene (e.g., ampicillin resistance) that allows researchers to identify cells that have taken up the plasmid by conferring resistance to an antibiotic.
- Origin of replication (ori): A sequence where DNA replication begins, allowing the plasmid to be copied within the host.
- Multi-cloning site (MCS) / Poly-cloning site: A short DNA sequence with multiple restriction enzyme sites that allow insertion of foreign DNA.
What is transformation?
The process of introducing foreign DNA (e.g., a plasmid) into a bacterial cell
What is a serial dilution of the plasmid?
A stepwise dilution of plasmid DNA to obtain a range of concentrations, often used to test how DNA concentration affects transformation efficiency.
What is LB?
Lysogeny Broth – a nutrient-rich medium used to grow bacteria.
What is E. coli?
A common bacterium used as a host for molecular cloning due to its well-understood genetics and ease of manipulation.
Purpose of each step in transformation:
- CaCl₂ incubation: Calcium ions help make the bacterial membrane permeable to DNA by neutralizing charges.
- Keeping the cells cold: Stabilizes the membrane and prepares it for heat shock.
- Adding the plasmid: Introduces the DNA you want the bacteria to take up.
- Heat shocking the cells at 42°C: Creates a thermal imbalance that allows the DNA to enter the cell.
- Adding ampicillin to the plates: Selects for bacteria that have taken up the plasmid (only plasmid-containing cells survive).
Difference between organismal and molecular cloning:
- Organismal cloning: Producing genetically identical organisms (e.g., cloning a sheep).
- Molecular cloning: Copying specific DNA sequences in microorganisms.
- In this lab: You did molecular cloning.
What two problems does molecular cloning solve?
- Amplifies specific DNA sequences.
-Enables study or manipulation of genes (e.g., protein expression, mutagenesis)
Recombinant plasmid:
A plasmid that contains inserted foreign DNA.
Bacteria (E. coli):
The host organism used to replicate and express plasmid DNA.
CaCl₂:
Used to make E. coli cells competent to take up DNA.
Heat shock:
Facilitates the uptake of plasmid DNA.
Transformation:
The process of introducing plasmid DNA into bacteria.
Essential plasmid elements: Poly-cloning site (MCS), Origin of replication, Selectable marker (ampicillin resistance gene)
- Poly-cloning site (MCS): Allows insertion of target DNA using restriction enzymes.
- Origin of replication: Ensures plasmid is copied inside the host.
- Selectable marker (ampicillin resistance gene): Allows selection of transformed cells.
Transformants vs. Transformation Efficiency
- Number of transformants: Actual number of colonies that grow.
- Transformation efficiency: Number of colonies per μg of DNA used (a measure of how effectively cells were transformed).
Is ampicillin effective at killing non-transformed bacteria?
Yes. Non-transformed cells lack the resistance gene and die.
Does CaCl₂ make cells more competent?
Yes. It increases membrane permeability to DNA.
Do all CaCl₂ cells get transformed?
No. Only a small percentage of cells take up the plasmid.
What effect does concentration of plasmid DNA have on transformation efficiency?
Higher concentrations generally increase transformation efficiency up to a point, after which it may plateau or even decrease due to cell toxicity or DNA aggregation.
