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Labs Flashcards

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Biology 101 flashcards
1
Q

Define pUC derived

A

plasmid present in e.coli

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2
Q

Define T7promoter

A

Located downstream from FP promoter, allows T7 RNA polymerase in cell to maintain continous expression

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3
Q

Define 6xHis tag

A

A poly-histidine, tag provides metal binding site for purification through NTA-chelate affinity chromatography

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4
Q

Where is the 6xHis tag found

A

In frame with a sequence that includes; N terminal fusion peptide, initiation codon. All located upstream from the fluorescent protein

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5
Q

What is the ori-locus

A

Origin of replication

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6
Q

What does the ori-locus do

A

Allows plasmid to be independently replicated in host cell.

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7
Q

What is the importance of ampicillin resistance in the experiment

A

Allows transformed cells to reproduce in presence of ampicillin which destroys e.coli cells ability to create cell walls. Meaning only successful cells would grow.

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8
Q

Define transformation

A

Inserting foreign plasmids into a cell to alter the genetic makeup

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9
Q

Define plasmid

A

Circular strands of DNA from cytoplasm of bacteria and yeast cells usually

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10
Q

Define cloning vector

A

Plasmids modified to include foreign DNA that can be expressed in bacteria to produce proteins within the vector

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11
Q

Where does the DNA for plasmids come from

A

Fungi, Bacteria, Plants, animals

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12
Q

Why is transformation important

A

Useful for genetic engineering because it acts like a virus and can create specialized proteins

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13
Q

How is insulin made

A

Transformation

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14
Q

Define competence, and how it is achieved in this lab

A

Some cells that cannot pick up plasmids naturally must be made competent to absorb it, this lab uses chemical transformation.

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15
Q

Define neutralization of charges and explain how it was achieved

A

Calcium cations in a solution can neutralize the negative charges in the cell membrane and DNA. Calcium chloride and heat shock were used to make the cells competent, then they were placed on ice in a calcium solution to be neutralized.

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16
Q

Why was neutralization of charges done on ice

A

The cold slowed down membrane fluidity to make a more efficient neutralization

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17
Q

Define heat shock

A

A sudden increase and subsequent decrease in temperature, causing a change in pressure between the insideq and outside of the cell, forcing the plasmid in through the pores

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18
Q

How is it ensured that the colony is identical

A

One bacterial cell performer binary fission to produce the entire colony

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19
Q

Briefly explain how cell extract was prepared

A

1 isolated colony was inoculated in LB and ampicillin solution
The cells were allowed to grow as they are incubated under agitation
After cell growth cell membranes are broken with a lysis treatment and a detergent which release cytoplasm

20
Q

what detergent is used in this lab

A

SDS

21
Q

What is LB

A

A nutritious broth to aid growth

22
Q

What does agitation do

A

Provides cells with oxygen, causing faster growth and division

23
Q

Briefly explain NTA-Chelate affinity chromatography

A

The 6x-His tag in fluorescent proteins binds to nickel in the nickel charged affinity resin, which prevents them from washing away like non fluorescent proteins

24
Q

4 steps for NTA-Chelate affinity chromatography

A
  1. Lysis of membrane
  2. Addition of nickel charged resin
  3. Washing resin for non bound components
  4. Elution of protein using elution buffer
25
Q

What elution buffer is used in this lab and why

A

Imdiazole, it has a high affinity for nickel

26
Q

Define Buffer A

A

50mM monobasic sodium phosphate, 300mM NaCl, 10mM imidazole at pH 8, adjusted by NaOH

27
Q

What was buffer A used for

A

Used to rinsecellular components without 6xHis tag that did not bind to nickel resin, leaving behind nickel resin and recombinant proteins

28
Q

Define maximum excitation value

A

The wavelength of light most readily absorbed by a fluorescent protein and results in the most fluorescence

29
Q

What lab did we collect maximum excitation value

A

Lab 2

30
Q

What lab quantified the protein

A

Lab 3

31
Q

What absorption spectrum was used, and what interval

A

visible light spectrum, 400-700nm at a 2nm interval

32
Q

What operates as the blank

A

The maximum excitation of the buffer

33
Q

What are the components of a spectrophotometer

A

Light source
Collimator
monochromator
wavelength selector
solution, in cuvette
detector
display

34
Q

Define bradford assay

A

Staining proteins with a dye then running it through spectrophotometry to determine the amount of light that was absorbed by the solutionH

35
Q

How does absorption relate to concentration

A

Absorption linearly rises with increase in concentration, only before absorption rates of 2

36
Q

What dye is used in bradford assay

A

Coomassie blue G-250, turns green to blue when in presence of small proteins

37
Q

What are the 3 sources of absorption when light passes through a solution

A
  1. Container
  2. Solvent
  3. Dissolved molecules
38
Q

Define the standard curve

A

Line of best fit for known concentrations (x) vs absorption spectrum (y)

39
Q

What does SDS-PAGE mean

A

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

40
Q

What is SDS-PAGE

A

All proteins are made negative from SDS and then ran through PAGE as they will be attracted to its positive side and repulsed from the negative. The gel will stop proteins based on weight at varying molecular weight markers

41
Q

In SDS-PAGE do heavier or lighter proteins go further

A

Lighter

42
Q

Why do we used LogMW

A

Migration distance decreases exponentially when molecular weight increases, and a log value will produce a more linear relationship with migration distance

43
Q

What is SDS used for in the lab

A
  1. Keeps the proteins denatured in linear chains
    2.Ensures a uniform negative charge on all proteins
    overall ensures only the weight effects migration
44
Q

Dimer vs Tandem Dimer

A

Tandem dimers are not split into subunits by denaturation, and are held together by strong covalent bonds and are not naturally occuring

45
Q

4 Characteristics of pRSET-FP plasmid

A
  1. 6xHIS tag
  2. T7 promoter
  3. Ampicillin resistance
  4. the plasmid codes for the FP production
46
Q

Calculate DF

A

solute/solution total

47
Q

A570 means absorbance at 570

A

Remember to subtract blank

cell bio labs (1 decks)

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