Lab 8-Plasmid Isolation

Question 1

What is Centrifugation?

Answer 1

Centrifugation is the process of separating substances based on their size and density by applying a centrifugal force.

Question 2

How centrifugation is performed in the lab ?

Answer 2

• a centrifuge is used to spin tubes containing a mixture of substances at high speed to collect dense components at the bottom of the tube while less-dense components remain in suspension.
• This rotor then spins at a controlled speed, applying centrifugal force to the tube, separating components based on sedimentation principles.

Question 3

What is pellet and supernatant ?

Answer 3

The collected particles that settle at the bottom of the tube are referred to as the pellet while materials that remain in suspension are referred to as the supernatant

Question 4

What does the level of separation depends on ?

Answer 4

The level of separation of a mixture depends on the speed. At low speeds, only very large substances will be pelleted. For example, cells in suspension. At higher speeds smaller particles may be pelleted, for example, nucleic acids. Therefore by controlling the speed over a series of processes or steps, it becomes possible to separate very specific components of a mixture.

Question 5

Centrifuges MUST be

Answer 5

balanced
It is critical to balance centrifuges across the centre of the rotor.
This means it is sometimes necessary to load an additional tube, often filled with water to even out the load. This tube is referred to as a ‘balance’.
Tubes should fit securely into the rotor so they do not move around during the run. Tubes also have a maximum speed that they can be subjected to

Question 6

Clean up Spills

Answer 6

Chemical agents can be corrosive to the rotor or internal part of the centrifuge. This comprises it making it dangerous as well as damaging expensive equipment.
Biological spills need to be cleaned up for contamination reasons

Question 7

Plasmid Mini Preparation

Answer 7

In relation to plasmid handling in the laboratory, the term ‘preparation’ is used to describe isolated, purified plasmid DNA from bacterial cells. A mini-preparation indicates a small-scale preparation, typically containing up to 50 µg of DNA.

Question 8

There are several methods that can be used to isolate DNA:

Answer 8

Ethanol precipitation
Phenol-chloroform extraction
Spin-column

Question 9

main steps in isolation of DNA

Answer 9

Growth → Lysis → Removal of Genomic DNA→ Purification

Question 10

alkaline lysis

Answer 10

a method of lysing cells in an alkaline solution such as NaOH.

Question 11

spin column purification

Answer 11

A spin column is a small inert that sits inside a 1.5 mL collection or Eppendorf tube (Figure 3). As the name suggest, this insert has a column shape and a small silica membrane at the bottom that liquid can pass through. Solution containing DNA can be pipetted into the column and centrifuged. The centrifugal force pushes the solution through the membrane. During this process however, DNA binds to the silica membrane and is subsequently separated from the solution. Then after a series of washes, DNA is released in a purified form, ready for downstream application.

Question 12

Resuspension Buffer

Answer 12

Contains:
RNase A
Digests RNA once cells are lysed in the next step