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Molecular Biology Lab. > Lab 3. > Flashcards

Lab 3. Flashcards

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1
Q

What are the 3 steps that are involved in the isolation and quantification of plasmid DNA from bacteria?

A

The preparation and removal of a bacterial lysate.

The adsorption of DNA on to a silica membrane.

The washing and elution of plasmid DNA.

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2
Q

What are plasmid minipreps used for?

A

As a method to recover plasmid DNA that has been replicated within bacterial cells.

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3
Q

Why would scientists want to replicate plasmid DNA within bacterial cells?

A

So they amplify and utilise specific DNA sequences.

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4
Q

What is the most popular and cost-effective method for isolating plasmid DNA from bacteria?

A

The silica gel spin column.

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5
Q

What is the plasmid miniprep procedure based on?

A

On the alkaline lysis of bacterial cells.

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6
Q

During the miniprep, what is alkaline lysis of bacterial cells followed by?

A

By the absorption of DNA onto a silica gel under high salt conditions.

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7
Q

Bacteria are lysed under what kind of conditions?

A

Under alkaline conditions.

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8
Q

What is the lysate?

A

The products that are removed from the bacterial cell after lysis.

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9
Q

What happens to the bacterial lysate after it has been removed from the bacterial cell under the alkaline conditions?

A

It is neutralized and changed to high-salt binding conditions.

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10
Q

Why is the lysate centrifuged after it has been adjusted to the high-salt binding conditions?

A

So that any bacterial cell debris can be removed.

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11
Q

What solution can be used to tell if complete bacterial lysis has occurred?

A

Lyse Blue.

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12
Q

How does Lyse Blue indicate complete bacterial lysis has occurred?

A

A blue solution will be formed if complete bacterial lysis has occurred.

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13
Q

What happens to the blue solution that is formed by Lyse Blue during the centrifugation step?

A

The solution will turn into a clear lysate or supernatant.

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14
Q

How do the silica membranes absorb plasmid DNA?

A

They selectively absorb plasmid DNA in high-salt buffers and they will remove plasmid DNA in low-salt buffers.

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15
Q

What in this experiment ensures that only DNA will be absorbed into the silica membrane?

A

The optimized buffers in the lysis procedure, combined with the selectivity of the silica membrane.

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16
Q

Where will the RNA, cellular proteins or metabolites from the bacterial cell be found if they are not present on the silica membrane?

A

They will be found in the flow-through.

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17
Q

Why are endonucleases removed during the silica membrane washing steps?

A

To prevent degradation of the plasmid DNA.

18
Q

How is the salt from the high salt buffer removed from the DNA sample?

A

In the washing steps.

19
Q

How is the purified plasmid DNA eluted from the silica membrane column?

A

Through the use of a low-salt buffer or water.

20
Q

What is the efficency of the elution of purified plasma DNA dependent on?

A

On pH.

21
Q

At what pH will the best eultion of purified plasma DNA take place?

A

Between pH 7.0 and pH 8.5.

22
Q

What is the average yield of DNA from 1.5 ml of bacterial culture?

A

Between 5 ug and 15 ug of plasmid DNA.

23
Q

What does the P1 re-suspension buffer contain?

A

Contains RNase A.

Contains LyseBlue.

24
Q

Is Lyse Blue essential to performing successful plasmid elutions?

A

No.

25
Q

What should an emperimenter do if they are using Lyse Blue and their suspension contains localized regions of colorless solution or if brownish cell clumps are visible?

A

They should continue mixing the solution until a homogenous blue suspension is observed.

26
Q

What was found in the P2 alkaline lysis buffer?

A

It contains NaOH/SDS in the presence of RNase A.

27
Q

What is the function of the SDS in the P2 alkaline lysis buffer?

A

It makes the phospholipid and protein components of the cell membrane soluble and this leads to cell lysis.

28
Q

What is the function of the NaOH in the P2 alkaline lysis buffer?

A

The alkaline conditions created by NaOH denature the proteins as well as the chromosomal and plasmid DNAs.

29
Q

What indicates that the SDS from the lysis buffer has been effectively precipitated after the N3 neutralisation buffer has been added?

A

The formation of a colorless solution.

30
Q

What does the high salt concentration of the N3 neutralisation buffer allow for?

A

For the precipitation of denatured proteins, chromosomal DNA, cellular debris, and SDS upon centrifugation.

31
Q

What will the plasmid DNA do after the addition of the N3 neutralisation buffer?

A

It will renature and stay in solution.

32
Q

How must the solution be mixed to ensure that complete precipitation of cell debris to occur?

A

It must be gently mixed and not vortexed.

33
Q

Where will plasmid DNA be found after mixing the cell debris?

A

It will be found in the clear supernatant, whereas the cell debris will form a pellet at the bottom.

34
Q

Why is vortexing the cell debris not a good idea?

A

Because it will shear the bacterial chromosome which will leave free chromosomal DNA in the supernatant.

35
Q

What are the silica gel membranes used for in lab 3?

A

For the selective adsorption of DNA in high-salt buffer.

36
Q

What happens during the washing steps of the experiment?

A

Endonucleases and are removed the high-salt buffer.

37
Q

How is plasmid DNA eluted from the silica membrane?

A

Through the use of a low-salt buffer or water.

38
Q

What is the absorbance of the nitrogenous bases during mass spectrometry?

A

260 nm.

39
Q

What is the equation to work out DNA concnetration after mass spectrometry?

A

DNA concentration (µg/mL) = (OD 260) x (dilution factor) x (50 µg DNA/mL)/ (1 OD260 unit)

40
Q

What is the maximum absorbance of proteins during mass spectrometry?

A

280 nm.

41
Q

What causes the high absorbance of proteins during mass spectrometry?

A

The tryptophan residues.

42
Q

How does absorbance measure the purity of a DNA sample?

A

The closer the sample is to 260 nm the purer the sample.

Molecular Biology Lab. (7 decks)

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