Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

methods > Midterm 1 > Flashcards

Midterm 1 Flashcards

1
Q

what is the goal of gene cloning

A

to multiply the number of copies for expression. copies must be identical

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What do you need to do to the gene prior to cloning?

A

purify it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Define gene

A

A piece of DNA/ segment of DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are 3 things a gene should have?

A

Promoter, ORF, and terminator for translation to occur and protein to be expressed.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Where does RNA polymerase bind

A

to the promoter

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What does an ORF have?

A

Start codon, stop codon

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Name the start and stop codons

A

Start: ATG Stop: TAG, TGA, TAA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How many ORFs does a gene have?

A

1

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How many ORFS does a DNA strand have? Are all true?

A

6; No only 1 is

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Where does translation start and stop

A

Start: Start codon. Stop: Stop codon.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Where does transcription happen?

A

the terminator

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are genes translated into a protein called?

A

Structural genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Why can you only take the ORF not the promoter in gene cloning?

A

The promoter is where the RNA polymerase binds. There are different types of RNA polymerase therefore you may not be able to get a functional gene because the used RNA polymerase does not match the promtoer and foes not bind. Therefore translation cannot happen.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Define DNA sequencing

A

Techniques enabling the structures of individual genes to be determined

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are PCR temperatures determined by?

A

The length of fragments and the number of GC.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are the number of hydgrogen bonds between AT and GC?

A

2 and 3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Where does Taq Polymerase come from

A

Thermos aquasis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

How do you determine the number of fragments given by PCR

A

2^ number of cycles

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Who invented PCR?

A

Kary Mullis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

State the steps of gene cloning.

A

1.A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule.
2 The vector transports the gene into a host cell, which is usually a bacterium, resulting in a bacterium carrying the recombinant DNA molecule
3 Within the host cell the recombinant DNA multiplies.
4 The host cell divides, passing down the recombinant DNA molecule.
5 Numerous cell divisions occur resulting in colonies of identical host cells. Each cell in the clone contains one or more copies of the recombinant DNA molecule. The gene carried by the recombinant molecule is now said to be cloned.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

How is PCR different from Gene cloning?

A

Rather than a series of manipulations involving living cells, PCR is carried out in a single test tube simply by mixing DNA with a set of reagents and placing the tube in a thermal cycler, a piece of equipment that enables the mixture to be incubated at a series of temperatures that are varied in a preprogrammed manner.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What are the 3 steps of PCR?

A

Denature, annealing, extension

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What occurs at denaturing, include temperature.

A

Mixture is heated to 95 C and hydrogen bonds between nucleotide on opposite strands break.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What happens after denature, before annealing? Why don’t the strands join back together?

A

The temperature is cooled down to 50-60C allowing the primer to attach. The two strands do not join together because there is an excess amount of primers.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What happens in extension.

A

Temperature is raised to 72C, the ideal condition for taq polymerase to work. The polymerase attaches to one end of each primer and synthesizes new strands of DNA complementary to the DNA.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What indicates success in a gene cloning experiment?

A

Identification of the gene of interest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

How are the regions of the starting DNA molecule marked?

A

The annealing positions of the primers.

28
Q

What are the two limitations of PCR.

A
  1. Must know sequence of DNA to design primers who can anneal. If the sequence is unknown, makes it difficult to design primers.
  2. Length of DNA sequence that can be copied is limited.
29
Q

How do we isolate long genes or genes that have never been studied?

A

Gene cloning

30
Q

What else can be done if the sequence of the gene is nor known?

A

Look at what is known about the sequence of an equivalent gene.

31
Q

True or false: PCR has good sensitivity?

A

True

32
Q

Define plasmids. Can they replicate independently of a host.

A

Small circle of DNA found in bacteria. Yes

33
Q

Name and define 3 types of DNA that can be used for gene cloning

A

Total Cell DNA: required as a source of material from which to obtain genes to be cloned
Plasmid DNA: must be separated from the main bulk of chromosomal DNA also present in the cell.
Phage DNA: needed if a phage cloning vector is to be used. Phage DNA is generally prepared from bacteriophage particles rather than from infected cells, so there is no problem with contaminating bacterial DNA.

34
Q

What are the steps of total DNA preparation from a culture of bacterial cells

A

1 A culture of bacteria is grown and then harvested.
2 The cells are broken open to release their contents.
3 This cell extract is treated to remove all components except the DNA.
4 The resulting DNA solution is concentrated

35
Q

Name and Define the two typical growth mediums.

A

Defined: All components and functions are known.
Undefined: Precise identity and quantity of components are not known

36
Q

Name all the components of LB broth and what they do.

A

Tryptone supplies amino acids and small peptides
yeast extract (a dried preparation of partially digested yeast cells) provides the nitrogen requirements.
sugars and inorganic and organic nutrients.

37
Q

Define chemical lysis

A

involves one agent attacking the cell wall and another disrupting the cell membrane.

38
Q

What do EDTA and SDS do in cell disruption

A

EDTA removes magnesium ions that are essential for preserving the overall structure of the cell envelope, and also inhibits cellular enzymes that could degrade DNA.
Detergents aid the process of lysis by removing lipid molecules and thereby cause disruption of the cell membranes.

39
Q

How can DNA concentration be measured? At what absorbance?

A

ultraviolet (UV) absorbance spectrophotometry.
260nm

40
Q

True or False

A

The amount of UV radiation absorbed by a solution of DNA is not directly proportional to the amount of DNA.

41
Q

where does a gene originate from?

A

genome

42
Q

Define conformation

A

the overall spatial configuration of the molecule, with the two simplest conformations being linear and circular.

43
Q

Which is lighter and less dense DNA or protein?

A

protein

44
Q

How does ethidium bromide work?

A

binds to DNA molecules by intercalating between adjacent base pairs, causing partial unwinding of the double helix.

45
Q

Why can the yield of a bacterial culture be low?

A

plasmids make up only a small proportion of the total DNA in the bacterial cell.

46
Q

How can you increase the amount of plasmid DNA?

A

Plasmid amplification

47
Q

What are good features of DNA to be cloned?

A

must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be produced and passed
relatively small, ideally less than 10 kb in size, as large molecules tend to break down during purification, and are also more difficult to manipulate.

48
Q

What are two DNA molecules that are good for cloning?

A

plasmids and bacteriophage chromosomes.

49
Q

What are two important factors in a plasmid used for cloning?

A

The size and copy number

50
Q

Define copy number

A

number of molecules of an individual plasmid that are normally found in a single bacterial cell.

51
Q

What is the size range of plasmids?

A

1.0 kb for the smallest to over 250 kb for the largest plasmids.

52
Q

how are genes of antibiotic resistance in plasmids used in the lab?

A

As a selectable marker to ensure the bacteria contains the cloned plasmid.

53
Q

What are the two names for copy number in plasmids?

A

Stringent - low copy number
Relaxed - high copy number

54
Q

What are the two groups plasmids fall into?

A

Conjugative
Non conjugative

55
Q

Why should bacterial liquid cultures be shaken?

A

to ensure aeration and oxygen and nutrient availability as well as to avoid bacterial settlement on the flask bottom which would result in cell death from the lack of nutrient availability.

56
Q

How large is the human genome?

A

3.2 billion base pairs resulting in 20-25,000 genes

57
Q

Define genome

A

The complete set of nucleotides.

58
Q

How big is commamonas testosterone>

A

0.78kb

59
Q

What are two strategies of cloning?

A

Library based cloning
PCR based

60
Q

When can you used PCR Based?

A

When you know the sequence of the gene of the amino acid sequence of the protein.

61
Q

Name the steps in gene cloning

A

Isolation, Insertion, Amplficiation, selection, confirmation

62
Q

What are the 7 principles of primer design?

A

15 to 20 bp
0 to 60% GC content
Start and end with 1 to 2 GC pairs
Melting temperature (™) :60 - 70 C
Primer pairs (FP & RP) -should have temperature within 5C of each other
Primers should not have complimentary regions
3 end of the primer must completely correspond to template DNA strand

63
Q

Define melting temperature and state the formula

A

Melting temperature: Temperature at which the strands separate.
™ = 4(G+C) + 2 (A+T) (Not used in real life)

64
Q

True or false: If you know the melting temperature you can determine annealing temperature. How much does the annealing temperature need to be.

A

True; 5 degree less otherwise primers will not anneal.

65
Q

Why is it important Primer pairs have a melting temperature of maximum of 5 degrees apart?

A

If the temperature is too difference one primer may not anneal at the annealing temperature.

66
Q

Why should primers not have complimentary regions?

A

Because they can bind to each other if they do.

methods (14 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions