Exam 1 - GP

Question 1

LB (Luria Broth)

Answer 1

A solution or agar made without any antibiotic

Used as a control

Question 2

Ampicillin (Amp)

Answer 2

An antibiotic that prevents bacterial growth

An irreversible inhibitor of the bacterial enzyme transpeptidase

Bacteriostatic; does not kill bacteria but prevents them from growing larger

Allows microsatellites to grow in the presence of Amp

Question 3

Transpeptidase

Answer 3

Used by bacteria to make their cell walls (cross-linking peptidoglycan chains)

Inhibited by ampicillin

When bacteria are exposed to ampicillin, the cell walls in bacteria cannot grow

Question 4

Plasmid

Answer 4

A small, circular dsDNA molecule found in bacteria

Are additional pieces of DNA that are different from the bacteria’s genomic DNA

Extrachromosomal DNA element

Self-replicating; replicate independently of chromosomal DNA

Can move in and out of bacteria and often contain antibiotic resistance genes

Question 5

Microcentrifuge

Answer 5

a machine that spins samples

Question 6

Vortex

Answer 6

an instrument used to rapidly shake/vibrate the liquid in the tube

Question 7

Kanamycin

Answer 7

Bactericidal: kills bacteria via inhibiting translation

Interacts with the 30S subunit of prokaryotic ribosomes

Three mechanisms of resistance have been recognized:
1. Ribosome alteration
2. Decreased permeability
3. Inactivation of the drugs by aminoglycoside modifying enzymes

Question 8

X-gal

Answer 8

A substrate for the enzyme β-galactosidase

Produces blue color if the bacteria took up the plasmids
See blue → took up plasmid

Gets cleaved by B-galactosidase to produce blue color

Question 9

What is DNA ligation?

Answer 9

Takes two different plasmids and ligate them together

Question 10

What does DNA ligase do?

Answer 10

Fixes the phosphodiester bonds on the outside of the plasmid membrane

Catalyzes the formation of a phosphodiester bond between juxtaposed 5’ phosphate and 3’ hydroxyl termini in duplex DNA

Question 11

What would happen if you forgot to add DNA ligase to your ligation mixture and why?

Answer 11

They wouldn’t stick together; wouldn’t get a recombinant plasmid (point of experiment)

Question 12

In this experiment we essentially cut a gene (Kan resistance) from a plasmid and inserted it into the pAMP plasmid. What was the importance of cutting both plasmids with HindIII and BamHI. Why does this produce the desired fragment?
Purpose of using same restriction enzyme:

Answer 12

Have the same sticky ends to ligate together

Question 13

How did we make a 1% TBE agarose gel for DNA electrophoresis—what components were needed?

Answer 13

ABED:
Agarose (higher % → more stiff/rigid gel)
TBE buffer (10X stock solution diluted to 1X)
EtBr
DI water

Make a working solution of 1x TBE
Measure out 60 ml of 1xTBE with a graduated cylinder and pour into Erlenmeyer flask
Weigh out 0.6 g (600mg) of agarose
Microwave for 1 min.

Question 14

Blunt end cloning is more versatile but less efficient than sticky end cloning

Answer 14

Advantages: Do not need to have compatible or matching sticky ends

Disadvantages: Interaction with the vector is more transient but it is not stabilized by the hydrogen bonding associated with sticky ends

Question 15

Plasmid map

Answer 15

shows the positions of where consensus sequences are found for different restriction enzymes

Question 16

Ampicillin resistance gene (Amp^r)

Answer 16

A gene that is found on the plasmid.

Produces a protein that destroys the Ampicillin molecule.

Bacteria carrying the plasmid with the Ampr gene are resistant to the effects of ampicillin and can grow in its presence.

Question 17

DNA ladder

Answer 17

A series of DNA fragments with known sizes

Used to determine the size of DNA fragments in which the sizes are unknown.

We use this as our reference when performing agarose gel electrophoresis.

Question 18

DNA molecular weight standard

Answer 18

A series of known DNA fragment sizes that is used to determine the sizes of unknown DNA fragments.

Often called a DNA ladder

Question 19

Lambda (λ) Phage DNA

Answer 19

Linear DNA from the λ phage virus that infects bacteria

Phage viruses attack bacteria like E. coli

Easy to obtain

HindIII restriction enzyme digestion (cutting) of λ phage DNA is commonly used as a molecular standard

Question 20

Genomic DNA, regardless of the source, can be digested with restriction enzymes that recognize….

Answer 20

4-8 consecutive bases

As these recognition sites occur in the DNA, the enzyme will cut the DNA at the restriction enzyme site

Question 21

Restriction Enzymes

Answer 21

Also called endonucleases because they cleave phosphodiester bonds in DNA

Are isolated from specific types of bacterial strains and are believed to have evolved as a defense mechanism

Proteins that bind to consensus sequences. Upon binding, the RE makes a double-stranded DNA break/cut

Different RE’s bind to different DNA sequences.

Question 22

Gel Electrophoresis

Answer 22

The standard lab procedure for separating nucleic acid by size (e.g. length in base pairs) for visualization and purification.

Uses an electrical field to move the negatively charged DNA toward a positive electrode through an agarose gel matrix.

The gel matrix allows shorter DNA fragments to migrate more quickly than larger ones.

You can accurately determine the length of a DNA segment by running it on an agarose gel alongside a DNA ladder.

Question 23

Agarose

Answer 23

A polysaccharide polymer extracted from seaweed

When agarose forms a gel, it is porous. Pore size affects the size of the DNA that is sieved.

Lower concentration of agarose, larger pore size → Larger DNA fragments can be resolved (or sieved).
Higher concentration of agarose, smaller the pore size → Smaller DNA fragments can be resolved.

Typical concentrations of agarose gels are 0.8-2.0%

Question 24

Agarose Gel Electrophoresis

Answer 24

A method to analyze sizes of DNA fragments

Relies on casting a gel made with agarose, which serves as a sieve

DNA is loaded into small wells and a current is supplied such that DNA migrates through the agarose

A current can be used because DNA is negatively charged (phosphate backbone) and will migrate toward the positive charge.

The larger DNA fragments travel the slowest and stay toward the top of the gel (where the wells are). The smaller DNA fragments travel the fastest and move more rapidly toward the bottom.

Question 25

How do we see the DNA?

Answer 25

We add ethidium bromide (EtBr) to the gel. EtBr is an aromatic compound that is fluorescent. EtBr is excited under UV light and emits an intense orange light when bound to nucleic acid. EtBr is a known mutagen!

Question 26

Consensus sequence

Answer 26

The DNA sequence that the restriction enzyme finds, binds to, and “cuts” the DNA. The consensus sequence for HindIII is AAGCTT.

Question 26

Why do we need a TBE running buffer?

Answer 26

Ensures the electric current flows through the gel Maintains a stable pH, which is essential for proper DNA migration and separation

Question 27

Linear DNA

Answer 27

DNA that is not connected at either end. Think of a piece of string. Eukaryotic DNA (like ours) is linear Some viruses also have linear DNA Prokaryotic organisms (like bacteria) have circular DNA

Question 28

Isoschizomers

Answer 28

RE's that recognize and cleave the same DNA sequence, but originate from different sources, and may have different optimal reaction conditions or methylation sensitivities. EcoRI 5’ G/AATTC 3’ FunII 5’ G/AATTC 3’

Question 29

pBR322

Answer 29

A plasmid that contains many different RE sites and is used commonly in molecular cloning studies. Also referred to as a cloning vector

Question 30

Molecular cloning

Answer 30

The process of creating new DNA molecules for research and/or medical purposes Restriction enzymes are often an important step to this process

Question 31

When a restriction enzyme cuts the DNA and leaves sticky ends...

Answer 31

There are 5’ or 3’ DNA overhangs that are complementary If sticky ends are complementary, then DNA molecules can be attached with greater ease.

Question 32

When restriction enzymes cut the DNA leaving blunt ends...

Answer 32

There are no DNA overhangs It is possible to attach (ligate) DNA molecules with blunt ends, but the efficiency is greatly reduced

Question 33

Gel extractions

Answer 33

Used to isolate a desired DNA fragment from an agarose gel following gel electrophoresis Use gel electrophoresis to separate gene insert from the vector Commonly used in molecular cloning to isolate a particular fragment of DNA after a restriction digest or PCR By placing the agarose gel on a UV transilluminator, we can visualize the fragment, cut it out from the agarose gel, and use a gel extraction method to extract the DNA from the gel.

Question 34

In Plasmid Isolations, we want plasmid DNA from bacteria. What other components are in the bacteria that we do not want to isolate?

Answer 34

CRMP: Chromosomal DNA RNA Membranes Proteins

Question 35

Resuspension Buffer (R)

Answer 35

The first solution used in a plasmid mini-prep to break open bacterial cells and extract plasmid DNA. Components: Glucose; Tris (Maintains pH stability), EDTA (inhibits DNases, preventing DNA degradation) and RNAse An isotonic solution that will prevent DNA degradation. Contains RNAse, an enzyme that will degrade RNA so it doesn’t contaminate the plasmid DNA.

Question 36

Lysis Buffer (L)

Answer 36

Breaks open bacterial cells and releases their contents, including plasmid DNA. Contains: 1. SDS (Sodium Dodecyl Sulfate): A detergent that dissolves the bacterial membrane by disrupting lipids and proteins. Denatures proteins, including nucleases that could degrade DNA. 2. NaOH (Sodium Hydroxide): Alkaline environment denatures both plasmid and genomic DNA. Disrupts hydrogen bonds between DNA strands, making the DNA single-stranded.

Question 37

Precipitation Salts (Psalts)

Answer 37

Used in the neutralization step of plasmid mini-prep to help separate plasmid DNA from cellular debris, including genomic DNA and proteins. Potassium acetate/Acetic acid Low pH; the alkaline lysis part of the procedure Neutralizes the NaOH so macromolecules begin to re-nature Large macromolecules (Chromosomal DNA, Lipids and Proteins) form non-specific interactions which creates large insoluble aggregates (white cloudy stuff) Small macromolecules (plasmid DNA) renatures correctly and are soluble in solution

Question 38

Silica columns/Column clean up

Answer 38

Result after alkaline lysis step is very “dirty” plasmid DNA: Lots of salts; EDTA; RNAse; cellular proteins and membranes Now we clean it up using the column purification Binding buffer: Salts that denature the DNA: Under these conditions the DNA will selectively bind to the silica resin but the rest of the material (cell debris, protein etc.) cannot bind. Wash (EtOH) a few times to get rid of the contaminants. Add a low salt buffer (DNA will renature) and will lose affinity for the resin. DNA is eluted (Remove (an adsorbed substance) by washing with a solvent)

Question 39

Plasmid Isolation steps:

Answer 39

1. isolate plasmids from bacteria 2. digest (cut) 3. ligation to create new plasmids

Question 40

Alkaline Lysis Steps:

Answer 40

1. Plasmid Isolation Pellet bacterial culture and add resuspension buffer to prevent DNA degradation. Resuspension buffer contains RNAse so all RNAs will be degraded. 2. Lysis buffer contains a detergent SDS which will dissolve lipid components (cell membranes) and lyse the bacterial cells. Enables the nucleic acids to be in solution. The high pH of the lysis buffer denatures proteins and causes DNA to become single-stranded. 3. Add precipitation salts have a low pH, which neutralizes the high pH of the lysis buffer. Large macromolecules (chromosomal DNA, lipids and proteins) form non-specific interactions which creates large insoluble aggregates (the white, cloudy gelatinous pellet). Small macromolecules, like plasmid DNA, renature correctly and is soluble in solution (found in the supernatant).

Question 41

The goal of plasmid isolations (or mini-preps) is to....

Answer 41

isolate and purify plasmid DNA from bacteria

Question 42

Column Steps: Purification

Answer 42

1. Binding buffer (added so the plasmid DNA can adhere to the silica membrane on the columns) 2. Wash buffer (contains EtOH, is added to help clean the DNA of contaminants) When a low salt solution, like water or TE buffer, is added to the column, the plasmid DNA will release from the membrane and the DNA is eluted.

Question 43

Ampicillin Resistance

Answer 43

Acquires gene that encodes b-lactamase Cleaves b-lactam ring of ampicillin When ampicillin is cleaved, it can no longer inhibit transpeptidase It behaves as a bacteriostatic drug in our experiments: Slows growth but does not kill bacteria Shown by microsatellite colonies

Question 44

Amp vs Kan

Answer 44

Ampicillin INHIBITS new cell wall synthesis; satellite colonies Kanamycin INHIBITS protein synthesis

Question 45

Why do you add loading dye to each sample? How much and why that amount?

Answer 45

To increase the density; helps sample sink into the wells and makes it denser than the buffer Not denser → spreads Aids in visualization; you can see it in the well and the current running through (confirms its moving in the right direction) Amount used: 6X stock solution concentration diluted down to 1X

Question 46

Factors that affect DNA mobility through an agarose gel:

Answer 46

% of agarose Time Size fragments of DNA (larger fragments stay up top) Electricity (higher voltage → faster movement) Shape (linear DNA vs plasmid)

Question 47

What conditions can you change in the gel to change how fast (or slow) the nucleic acid moves through the gel?

Answer 47

Current Concentration of the gel

Question 48

How do you resolve (more clearly see) two bands that are close in size?

Answer 48

Time: Letting things run longer; more time to separate Increase concentration of gel (ex. 1% to 2%)

Question 49

Describe the mechanisms by which restriction enzymes work

Answer 49

Based on palindromic sequences and restriction enzymes cutting them Staggered cut Leave single-stranded DNA overhangs at the ends of DNA fragments. Sticky ends; they can stick together by hydrogen bonds. Not staggered; blunt ends

Question 50

"Ingredients" to set up a restriction digest

Answer 50

1x Buffer (20ul reaction = add 2ul of 10x) 1/10 the final volume = volume of enzyme (this means that if you have a 20ul reaction you add 2 ul of enzyme. Remember enzymes are stored in glycerol so they do not freeze, but too much glycerol can inhibit reactions) Water (our variable): add to 20ul [or whatever sample size was] Example: (WEBD) Water: 15ul Buffer 1 (10x): 2 ul EcoR1: 2 ul DNA (1ug/ul): 1 ul Total volume: 20ul

Question 51

Name reasons why a restriction digest might not work.

Answer 51

Incorrect buffer; different buffers have different efficiencies Incubation period Temperature Understand the sequence you’re cutting and if your enzyme can cut it or not

Question 52

How to figure out what type of enzyme you would use to excise a gene fragment from a plasmid

Answer 52

1. Identify the Gene’s Location in the Plasmid 2. Select enzymes that flank (cut before and after) the gene without cutting inside it. 3. Choose an enzyme that works efficiently in standard buffer conditions.

Question 53

How do you perform a gel extraction?

Answer 53

Need to know the sequence of the gene of interest Put the sample through the column, dilute the contents, run a flash gel to test whether or not extraction was successful

Question 54

What is the end product of the alkaline lysis procedure (mini preps)?

Answer 54

a plasmid

Question 55

Resuspenion buffer

Answer 55

resuspends pellet (initial pellet is bacterial cells, then we try to isolate plasmid from the cells)

Question 56

lysis buffer

Answer 56

lyses things open

Question 57

Precipitation salts

Answer 57

help to precipitate out the macromolecules (DNA, plasmid membrane, proteins) but leaves plasmids behind

Question 58

Why do you not get chromosomal DNA from an alkaline lysis procedure?

Answer 58

Its big and doesn’t come back together correctly (fried egg is still protein but clear stuff is opaque)

Question 59

Why do you not get proteins from an alkaline lysis procedure?

Answer 59

It doesn’t stain solution, psalts precipitate it out

Question 60

Why do you not get RNA from an alkaline lysis procedure?

Answer 60

We use RNase to get rid of it

Question 61

Why do you not get membranes from an alkaline lysis procedure?

Answer 61

It's a macromolecule (phospholipid bilayer, proteins, and carbs) that gets precipitated out by psalts

Question 62

What is the purpose of adding Magnesium and Calcium Chloride to the bacterial cells during this procedure?

Answer 62

To add positive charges; the DNA and lipid membranes have a overall negative charge so they naturally repel each other CaCl neutralizes the charges so the DNA could pass through the membrane

Question 63

Why do we perform a heat shock?

Answer 63

To make the membrane porous; allows plasmid to enter We did a heat shock for 2min then put it back on ice

Question 64

Transformation not efficient

Answer 64

Not all cells become chemically competent; only some are able to take up DNA. All you need is 1 to grow in group of hundreds or thousands

Question 65

****Why do we grow the bacteria for an hour after the heat shock?

Answer 65

[did this without presence of antibiotics] To give them time to transcribe and translate We need this hour for transcription and translation - why it’s important to incubate Put on antibiotics → bacteria will die because they don't have enough time to transcribe or translate the antibiotic resistant genes

Question 66

What are microsatellite colonies and why do they form?

Answer 66

Tiny satellite bacterial colonies that appear around larger bacterial colonies when grown on ampicillin-containing media They don’t carry the resistance gene but survive because of β-lactamase diffusion Bacteria that carry the ampicillin resistance gene produce β-lactamase, which breaks down ampicillin in the surrounding area. Over time, ampicillin in the agar around resistant bacteria gets degraded. Once enough ampicillin is degraded, some non-resistant bacteria (or bacteria with weaker resistance) start growing in the cleared zones.

Question 67

How do you know if the bacteria took up the plasmid?

Answer 67

Able to grow on selected media Ones that took up amp grow on amp Ones that took up kanamycin grow on kan

Question 68

Cells that take up (transform) pBlu turn what color? Why?

Answer 68

Blue; the plasmid contains the lacZ gene, which encodes β-galactosidase