Practicals Flashcards
What method is used to extract plasmid DNA from bacterial cells?
Miniprep
What is the definition of transforming bacteria?
The process of inserting plasmids into the bacterial cells.
What does transforming bacteria allow?
This allows genes of interest to be inserted and replicated within the cells through the growth of bacterial colonies.
Why are genes inserted into plasmids for antibiotic resistance?
Allows bacterial cells which have been transformed to be selected for, by growing them on a media containing an antibiotic.
Each colony on a plate containing the selective growth medium will…
have grown from a single transformed bacteria.
What will happen when single colony is removed and allowed to grow into a liquid cell culture?
Replicate the inserted plasmid and gene of interest.
In liquid cell culture what happens to the bacteria?
Bacteria is added to a tube containing growth medium, rather than being spread onto an agar plate.
Why do you need to count the number of bacterial cell colonies on the plates?
To estimate the frequency with which the bacteria develop a mutation that makes them antibiotic-resistant.
Why do we count colonies?
Each colony will have grown from a single bacterium and gives an estimate of the concentration of cells added to a plate.
Why is it important to know the volume of bacterial culture and the number of dilutions?
It will be possible to calculate the concentration of cells in the original culture.
What is meant by logarithmic dilutions?
Serial dilutions which decrease in concentration by the same factor.
Why are undiluted suspensions or solutions used?
used to produce a range of dilutions, usually so the original concentration can be calculated.
Give an example of a diluent?
Distilled water or a buffer solution (added to to make each dilution)
Why is it beneficial that dilutions increase by the same factor at each step?
Creates a broad range of concentrations.
What does miniprep involve?
This involves breaking open the bacterial cells, and then separating the plasmid DNA from the proteins, chromosomal DNA and other cellular material.
What is the difference in the pellet and the supernatant after centrifugation?
Pellet is the material that collects at the bottom of the tube, and the supernatant is the liquid that remains above the pellet in the tube.
Why is the bacteria culture centrifuged?
To collect the bacterial cells at the bottom of the tube.
What is Lysis?
The growth media (supernatant) is removed, and a detergent is added to the bacteria. This dissolves the cell membranes, breaking open the bacteria and releasing the cellular contents.
Why is a high salt concentration added to the extract?
causes the chromosomal DNA to become insoluble, so it precipitates. The plasmid DNA does not precipitate. Centrifugation of the extract collects the chromosomal DNA at the bottom of the tube as a pellet. The supernatant containing the plasmid is removed.
Why is an alcohol (isopropanol) added to the plasmid solution?
Causes the plasmid to precipitate.
Why is agarose gel electrophoresis used?
The plasmids inserted into transformed bacteria can be separated from other nucleic acids
What buffer is used for running the gel?
Tris-acetate-EDTA or TAE, with a pH of ~8.5. It is a common buffer for agarose gel electrophoresis and is suitable for short gel run times (<4 hours) and large pieces of DNA (>20 kb).
How are the DNA molecules separated in the gel?
applying an electrical field across the gel, which causes DNA (naturally negatively charged) to move towards the positively-charged anode. Smaller DNA molecules move through the gel faster, thus the DNA is separated according to size. Plasmids are smaller than the bacterial chromosomal DNA, for example, so move further within the gel.
How does voltage effect the run time of the gel electrophoresis?
A low voltage will mean the gel takes too long to run. We are separating large pieces of DNA (~4kb), therefore we need a relatively high voltage to move them. Gel density also affects run time.
