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BIO 111 > Practial 2 > Flashcards

Practial 2 Flashcards

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1
Q

What are the 3 basic steps to DNA Isolation?

A

1.Cell Lysis
2.Protein Precipitation
3.DNA Precipitation/Cleaning

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2
Q

Is there a strict protocol for DNA Isolation

A

No, many different protocols may apply, all depends on the tissue, organism, and purpose

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3
Q

2 types of DNA

A

Plastid-DNA in mitochondria and chloroplasts
Nuclear- DNA from cells nucleus

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4
Q

What happens during Cell Lysis

A

We get rid of membranes with a lysis buffer
Note: still a lot of macromolecules and proteins to get rid off*

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5
Q

What happens during Protein Precipitation

A

The removal of histones and other proteins

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6
Q

What ways to do protein precipitation exist

A
  1. Protease- which is an enzyme that will degrade proteins
  2. Protein precipitate solution- you add it to the cell and it helps precipitate clumps of all those proteins together
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7
Q

Why do histones have high priority?

A

Because they have a positive charge and will cancel out DNA which has a negative charge

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8
Q

What happens during DNA precipitation?

A

We get DNA to precipitate out of solution

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9
Q

What is done after DNA is isolated in DNA isolation?

A
  1. We test for yield/product
  2. Amplify a particular sequence to see if we were successful in our isolation. If there is lots of DNA, you may see a stringy, white precipitate
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10
Q

What are enzymes?

A

an organic catalyst/protein that speeds up metabolic reactions

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11
Q

What is energy of activation? (Eact)

A

-the initial energy investment required to have a reaction start/proceed

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12
Q

How do enzymes work?

A

By lowering energy of activation (Eact) levels. They make it so the reactants use/have to obtain less energy and therefore get products faster.

Without enzymes, there is no such thing as life

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13
Q

Catalized reaction

A

Reaction with enzymes

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14
Q

Uncatalized reaction

A

Reaction without enzymes

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15
Q

Substrate

A

The substance that an enzyme acts on/ fits in the enzyme

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16
Q

Active Site

A

A shallow depression or groove on an enzyme where the substrate fits

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17
Q

How can I tell the enzyme is working?

A

-check for production of products

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18
Q

What are some factors that affect enzyme activity?

A

1.Time
2.Enzyme concentration
3.Temp
4.pH

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19
Q

how does time affect enzyme activity?

A

more time = enzyme activity increases

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20
Q

how does enzyme concentration affect enzyme activity?

A

if it increases, enzyme activity increases

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21
Q

how does temperature affect enzyme activity?

A

boiling=denatured, the lower the temp, enzyme activity increases

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22
Q

how does pH affect enzyme activity?

A

if pH is outside neutrality, enzyme is denatured. Must remain neutral

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23
Q

Is it possible for an enzyme to return to its natural state after being denatured?

A

Yes, occasionally whenever you remove the denaturing agent its possible

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24
Q

what happened when the enzyme/potato reacted with H2O2 in an environment w ph of 7

A

lots of bubbles appeared, indicating enzyme is working

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25
Q

what happened when the enzyme/potato reacted with H2O2 in an environment w ph of 2

A

no bubbles, indicates no product

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26
Q

what happened when the enzyme/potato reacted with H2O2 in an environment w ph of 12

A

no bubbles, indicates no product

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27
Q

What are the phases of interphase?

A

-G1
-S
-G2

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28
Q

What takes place in the G1 phase of interphase?

A

Increase in ribosomes and mitochondria as well as an increase in the size of the cell

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29
Q

What takes place in the S phase of interphase?

A

DNA replication and histones replicate; histones use the ribosomes/proteins and the DNA replication makes use of the atp from mitochondria

30
Q

What takes place in the G2 phase of interphase?

A

-Further increases in size
-Final Preparations for division
-produces 2 copies of DNA

31
Q

Can interphase be seen under a microscope?

A

No

32
Q

What takes place during prophase?

A

The chromosomes condense and the nuclear membrane starts to break down

33
Q

What takes place in metaphase?

A

Sister chromatids line up along equator

34
Q

What takes place in anaphase?

A

Cohesions are cleared/seperated by enzyme called separase
Sister chromatids separate and move along the kinetochore microtubules towards opposite ends of the cell.

35
Q

What takes place in telophase?

A

It’s the opposite of prophase. Chromosomes begin to unwind and nuclear membrane reforms.
We are left with 2 identical sets of chromosomes and 2 nuclei began to form into the cells nucleus

36
Q

Can the cell cycle be complete without cytokinesis?

A

Yes, it will form a cell with 2 nucleus

37
Q

What takes place in cytokinesis?

A

Depends on the type of cell; animal or plant, but basically finally separate into 2 individual daughter cells

38
Q

How is cytokinesis accomplished in an animal cell?

A

The formation of a cleavage furrow which contracts/ tightens the microfilaments of the cell until it is pulled apart

39
Q

How is cytokinesis accomplished in a plant cell?

A

Vesticles containing cell wall material transport the material to the equator of the cell and start to a cell wall in the middle of the cell until completely and form 2 daughter cells

40
Q

Can prophase be seen in a microscope?

A

Yes, with the use of a stain. The stain stains for nuclear proteins, look for were the stain isn’t evenly distributed throughout the nucleus

41
Q

What is PCR

A

A way to amplify a targeted portion of DNA. Involves using varying temperatures

42
Q

How is PCR done?

A

Used to be done by hand, but now we use a machine called a Thermal Cycler

43
Q

Steps of PCR

A
  1. Denaturing
  2. Annealing
  3. Extension
44
Q

What is an amplicon?

A

The products of PCR

45
Q

When A is on one strand,…

A

It will form 2 H bonds with a T on the other strand

46
Q

When G is on one strand,…

A

It will form 3 H bonds with a C on the other

47
Q

In what case would we substitute T for U

A

When talking about RNA

48
Q

What is done during the denaturing step of PCR

A

The temp is raised to around 95°C, this separates the 2 strands of our template DNA

49
Q

What is done during the annealing step of PCR?

A

The temp is lowered to around 55°C, this allows your forward and backward primer to bind

50
Q

What is done during the extension step of PCR?

A

The temp is raised to around 72°C, this allows Taq polymerase to start where the primers are. It will read one strand and form a new complementary strand based of CBP

51
Q

How long do the steps of PCR run?

A

A specific period of time, anywhere from 30 secs to 2 mins

52
Q

Can the temperatures used during PCR vary?

A

Yes, depending go the number of factors

53
Q

How long is the initial denaturing of PCR?

A

2-10mins

54
Q

How many times are the basic steps of PCR done?

A

30-35x

55
Q

What is the final extension done for?

A

To allow enough time for TAC polymerase to bind to amplicons and finish any of the amplicons that haven’t been able to

56
Q

At what temperature does the thermal cycler keep the template DNA at once PCR is done?

A

4°C

57
Q

What materials are needed to perform PCR?

A

-Template DNA
-Primers
-Taq polymerase
-dNTPs

58
Q

What two materials make the “master mix”

A

Taq polymerase and dNTPs

59
Q

What is the primers job in PCR?

A

It targets the segment and dictates what specific sequence your amplicon is going to be/replicate

60
Q

What is the dNTPs’ job in PCR?

A

A nucleotide that has 3 phosphates used to form master mix

61
Q

Why is the “master mix” used?

A

It is ideal bc it reduces pipetting and risk of contamination, is convenient, saves time and preempts possible errors in mixin

62
Q

What is Taq polymerase and what is its job in PCR?

A

It is bacteria taken from bacteria found in high temperatures and it is an ingredient for “master mix”

63
Q

What is Gel Electrophoresis?

A

A way to separate nucleic acids or proteins, with a focus on DNA only.

64
Q

How does Gel electrophoresis work?

A

Subjects DNA to an electric field, causing it to migrate towards positive end and separates/filters DNA fragments as it migrated through gel

65
Q

Which fragments pass through the gel quicker; small or large?

A

Small fragments pass quicker through gel

66
Q

Overview of steps of Gel Electrophoresis

A

1.Have to make a gel (Casting a gel)
2.Assemble parts and pipette DNA into wells
3.Run gel
4. Resolve any imaging issues

67
Q

Is there a set type of compound and composition of compound that is used to make gel for gel electrophoresis?

A

No, many different compounds and composition of said compounds can be used

68
Q

What compound is commonly used during gel electrophoresis?

A

Agarose

69
Q

How would different concentrations of compounds affect the gel?

A

It can affect the type of separation we see and vary based on pore size

70
Q

How is DNA precipitated from cell in DNA precipitation?

A

Since DNA is polar because of it’s “phosphate backbone”, we can use ethanol, which is less polar, to help precipitate the DNA out the cell

71
Q

What do enzymes do?

A

they are involved in bond breaking and/or bond formation

72
Q

How do the kinetochore microtubules shorten?

A

They depolymerize at the end at which they’ve grabbed the each sisterchromatid and pull them apart

BIO 111 (5 decks)

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