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BIOL 226 > Cloning > Flashcards

Cloning Flashcards

1
Q

Before cloning what is required..

A

the DNA needs to be cut. up before being inserted into a vector

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2
Q

What cuts the DNA before cloning

A

Bacterial restriction enzymes

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3
Q

Where does the enzyme EcoRi cut

A

between G and A nucleotides of each strand

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4
Q

What is the difference between “sticky” end and “overhangs”

A

Sticky - they stick to complementary sequences

Overhangs - provides cohesive ends for ligation

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5
Q

What are restriction maps

A

a map of known restriction sites within a sequence of DNA

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6
Q

What is the benefit of a restriction map?

A
  • Provides information on the location of genes
  • Restriction maps reflect true physical distance unlike genetic maps
  • can help construct physical maps of the entire genome
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7
Q

What can restriction fragments be useful for?

A

They can be used as molecular markers that reveal a blueprint of an individual genomes

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8
Q

What is gene cloning

A

the isolation and amplification of a given gene

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9
Q

Steps in cloning a gene

A
  1. Cut the plasmid and the DNA with a restriction enzyme that will create identical ends
  2. Incubate the 2 DNA molecule in the presence of a ligase
  3. Transform into bacteria to propagate and amplify
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10
Q

What is the benefit of AmpR

A

can be used to detect the presence of a plasmid backbone - this gene will help make the bacteria grow

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11
Q

Ampicillin

A

helps to prevent bacterial growth

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12
Q

Can genes stay alive without AmpR

A

No, because they are unable to grow

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13
Q

What encourages the bacteria to take up plasmids/DNA

A

the heat shock -

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14
Q

True or False - When a bacteria dies they were able to take up plasmids and AmpR

A

False

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15
Q

Is bacteria able to grow in the presence of ampicillin and AmpR

A

Yes, bacteria will be able to go because AmpR can grow in an environment that contains the antibiotic ampicillin.

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16
Q

Purpose of Alpha complementation

A

to ensure cooling was successful or not

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17
Q

What needs to occur for the cell to become blue?

A

Blue is an unsuccessful con where DNA was not inserted into the peptides and X-gal was able to be metabolized (B-gal “eats” up X-gal

Beta-gal + X-gal = functional B-gal

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18
Q

What needs to occur for the cell to remain white?

A

This represents a successful cloning - DNA was inserted into alpha peptides and therefore the X-gal was not able to metabolized. Beta-gal did not cleave X-gal because it had the DNA

No functional Beta-gal

19
Q

What colour is the cell where there is a alpha complementation versus when there is not

A

Blue - alpha complementation (unsuccessful cloning) + white is when there isn’t alpha complementation (successful cloning)

20
Q

True or False - Beta-gal is functional when alpha complementation occurs

A

Yes true

21
Q

True or false - in blue colonies DNA was inserted

A

False, it is with the white colonies that DNA is inserted where there is no functional Beta-gal

22
Q

What is Southern blotting

A

a technique used to detect a specific DNA sequence in a blood/tissue sample

23
Q

Purpose of gel electrophoresis

A

Through gel electrophoresis molecules in a mixture are separated

24
Q

What pole do DNA molecules migrate towards during gel electrophoresis?

A

the positive anode because of its original negative charge

25
Q

What determines the speed of migration

A

smaller fragments move easier and large are slower

26
Q

What makes bands visualized (stain)

A

Ethidium bromide

27
Q

What state does the DNA have to be in before proceeding with southern blotting

A

denatured

28
Q

Purpose of PCR

A

Used to quantify DNA in a region of interest between 2 primers

29
Q

What are the 3 steps of PCR?

A
  1. Denaturation
  2. Annealing
  3. Extension
30
Q

What occurs in the step “Denaturation” in PCR?

A

Temperature is raised to 90 degrees - this breaks the H-bonds between base pairs and releases 2 single strands

31
Q

What occurs in the step “Annealing” in PCR?

A

The temperature drops to 40-60 degrees and the short pieces of single stranded nucleotides sequences called primers are used to amplify to target DNA

32
Q

What occurs in the step “Extension” in PCR?

A

the temperature is increased to 72 degrees this is ideal for the tax DNA polymerase

33
Q

Purpose of Taq DNA polymerase

A

(DNA synthesis) synthesizes the first set of complementary strands by the addition of the four nucleotide triphosphate

34
Q

What is RFLP

A

Restriction Fragment Length Polymorphism - A technique that exploits homologous DNA sequences

35
Q

Benefit of using RFLP

A

used to distinguish individuals or pinpoint the location of specific genes within a sequence

36
Q

True or False - Using RFLP technique DNA can be extracted from individuals to map out how a mutation is migrating

A

True

37
Q

True or False: When cloning a large fragment it is the same steps as cloning other fragments

A

False - cannot use plasmids

38
Q

When cloning large fragment what type of vectors are required

A

Fosmids + BACs

39
Q

True or false: vectors in yeast and bacteria have high replicative power

A

No, false they have low replicative power but are able to carry larger DNA fragments

40
Q

What is gDNA

A

Whole-genome -> Genomic library - contains all the genes (introns and exons)

41
Q

cDNA

A

Only using exons that will be incorporated into mRNA (coding sequence)

42
Q

Hybridization

A

Hybridization allows us to extract and isolate DNA molecules to grow bacterial colony and amplify a desires gene

43
Q

True or False - with hybridization, you have non-affected bacterial cells and then you need to infect them to see the desired gene amplify?

A

True

44
Q

How is hybridization different than finding the clone of interest by using an antibody?

A

Antibody identifies specific plaques which are fusion proteins bound to the membrane while, hybridization uses bacteriophage to infect bacteria

BIOL 226 (6 decks)

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