Mini Prep Flashcards

1
Q

What is plasmid?

A

A double stranded, circular extra chromosomal DNA of bacterium

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2
Q

What is plasmid used for?

A

In recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA

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3
Q

What are the three steps of purifying plasmid DNA?

A

Growth of bacterial culture
Harvesting and lysis of bacteria
Purification of plasmid

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4
Q

How is alkaline lysis used?

A

Breaks the cells open to collect cell components

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5
Q

Outline the alkaline lysis method

A

Bacteria containing plasmid grown
Lyses with buffer containing SDS and NaOH
Through agitation, precipitation, centrifugation and the removal of supernatant, the plasmid is isolated

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6
Q

What does the SDS do in solution II?

A

Denatures cell proteins

Cleaves the phospholipid bilayer of the membrane

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7
Q

What does the NaOH do?

A

Disrupts hydrogen bonding between DNA bases which converts dsDNA to single stranded DNA
Also breaks down cell wall

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8
Q

Describe the re-suspension of bacterial pellet in alkaline lysis

A

Resuspend pellet in solution containing tris, EDTA, glucose and RNase A - normally called solution I

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9
Q

What are the roles of the components of solution I?

A

EDTA - chelates Mg+ and Ca2+ to stop DNAase damaged the plasmid. Also destabilises the cell wall

Tris HCl - buffer

Glucose - maintains osmotic pressure so cells don’t burst

RNAase A - degrade cellular RNA when cells are lysed

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10
Q

Explain the neutralisation with solution III in alkaline lysis

A

Addition of potassium acetate (solution III) to decrease the alkalinity of the mixture
plasmid DNA re-natures to dsDNA here
This dissolves in solution whilst everything else becomes a white precipitant

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11
Q

How can you separate precipitant from solution?

A

Centrifugation

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12
Q

Outline the cleaning and concentration in alkaline lysis

A

Clean out salts, EDTA, residual proteins, debris and RNase

Ethanol precipitation

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13
Q

What is LB broth?

A

Nutrient rich medium which is primarily used for growth of bacteria
Usually includes peptides; casein peptones; trace elements and minerals

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14
Q

How can bacteria be selectively grown?

A

Introduction of an antibiotic

In this instance it was kanamycin which was specific to the plasmid

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15
Q

What observation can be made with LB broth when bacteria successfully cultures?

A

Gets more cloudy as bacteria grows

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16
Q

Explain what the following component of the lysis buffer is for: tris hcl

A

Act as a buffer to maintain a stable pH

17
Q

Explain what the following component of the lysis buffer is for: Sucrose

A

Create an oncotic pressure

18
Q

Explain what the following component of the lysis buffer is for: EDTA, EGTA

A

Chelate divalent ions to prevent unwanted reaction

19
Q

Explain what the following component of the lysis buffer is for: sodium fluoride and sodium pyrophosphatase

A

Protease inhibitors to prevent enzymatic breakdown of the protein

20
Q

Explain what the following component of the lysis buffer is for: Triton X

21
Q

What are the components of the lysis buffer?

A
Tris HCl
Sucrose
EDTA
EGTA
Sodium fluoride 
Sodium pyrophosphatase
Triton X
22
Q

What are the advantages of using a lysis buffer?

A

Makes protein extraction simpler
Done without specialised equipment
Done at room temp on desk

23
Q

What are the disadvantages of using a lysis buffer at room temperature?

A

Room temp may cause degradation of the protein

24
Q

What does this method remove from the pellet and leave you with?

A

Removes protein and chromosomal DNA
then salts and small molecules at alcohol precipitation
Then leaves you with plasmid DNA

25
Explain the roles of the components of solution II?
NaOH - disrupts H bonding in the dsDNA bases to convert to ssDNA SDS - Detergent. Denatures proteins in cells and solubilises the membrane
26
Explain the roles of the components of solution III
K acetate - decreases alkalinity to allow renature of DNA Glacial - Ph balance Water - dsDNA can dissolve in water but genomic DNA cant. Helps denatured proteins clump together.
27
What can be used instead of isopropanol to washed the DNA pellet?
Phenylchloroform
28
Why is isopropanol used > phenylchloroform?
Nasty burns