Lab 3. Flashcards
(42 cards)
What are the 3 steps that are involved in the isolation and quantification of plasmid DNA from bacteria?
The preparation and removal of a bacterial lysate.
The adsorption of DNA on to a silica membrane.
The washing and elution of plasmid DNA.
What are plasmid minipreps used for?
As a method to recover plasmid DNA that has been replicated within bacterial cells.
Why would scientists want to replicate plasmid DNA within bacterial cells?
So they amplify and utilise specific DNA sequences.
What is the most popular and cost-effective method for isolating plasmid DNA from bacteria?
The silica gel spin column.
What is the plasmid miniprep procedure based on?
On the alkaline lysis of bacterial cells.
During the miniprep, what is alkaline lysis of bacterial cells followed by?
By the absorption of DNA onto a silica gel under high salt conditions.
Bacteria are lysed under what kind of conditions?
Under alkaline conditions.
What is the lysate?
The products that are removed from the bacterial cell after lysis.
What happens to the bacterial lysate after it has been removed from the bacterial cell under the alkaline conditions?
It is neutralized and changed to high-salt binding conditions.
Why is the lysate centrifuged after it has been adjusted to the high-salt binding conditions?
So that any bacterial cell debris can be removed.
What solution can be used to tell if complete bacterial lysis has occurred?
Lyse Blue.
How does Lyse Blue indicate complete bacterial lysis has occurred?
A blue solution will be formed if complete bacterial lysis has occurred.
What happens to the blue solution that is formed by Lyse Blue during the centrifugation step?
The solution will turn into a clear lysate or supernatant.
How do the silica membranes absorb plasmid DNA?
They selectively absorb plasmid DNA in high-salt buffers and they will remove plasmid DNA in low-salt buffers.
What in this experiment ensures that only DNA will be absorbed into the silica membrane?
The optimized buffers in the lysis procedure, combined with the selectivity of the silica membrane.
Where will the RNA, cellular proteins or metabolites from the bacterial cell be found if they are not present on the silica membrane?
They will be found in the flow-through.
Why are endonucleases removed during the silica membrane washing steps?
To prevent degradation of the plasmid DNA.
How is the salt from the high salt buffer removed from the DNA sample?
In the washing steps.
How is the purified plasmid DNA eluted from the silica membrane column?
Through the use of a low-salt buffer or water.
What is the efficency of the elution of purified plasma DNA dependent on?
On pH.
At what pH will the best eultion of purified plasma DNA take place?
Between pH 7.0 and pH 8.5.
What is the average yield of DNA from 1.5 ml of bacterial culture?
Between 5 ug and 15 ug of plasmid DNA.
What does the P1 re-suspension buffer contain?
Contains RNase A.
Contains LyseBlue.
Is Lyse Blue essential to performing successful plasmid elutions?
No.
