Exam 1 - GP Flashcards
LB (Luria Broth)
A solution or agar made without any antibiotic
Used as a control
Ampicillin (Amp)
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
Transpeptidase
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
Plasmid
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
Microcentrifuge
a machine that spins samples
Vortex
an instrument used to rapidly shake/vibrate the liquid in the tube
Kanamycin
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
X-gal
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
What is DNA ligation?
Takes two different plasmids and ligate them together
What does DNA ligase do?
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
What would happen if you forgot to add DNA ligase to your ligation mixture and why?
They wouldn’t stick together; wouldn’t get a recombinant plasmid (point of experiment)
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:
Have the same sticky ends to ligate together
How did we make a 1% TBE agarose gel for DNA electrophoresis—what components were needed?
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.
Blunt end cloning is more versatile but less efficient than sticky end cloning
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
Plasmid map
shows the positions of where consensus sequences are found for different restriction enzymes
Ampicillin resistance gene (Amp^r)
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.
DNA ladder
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.
DNA molecular weight standard
A series of known DNA fragment sizes that is used to determine the sizes of unknown DNA fragments.
Often called a DNA ladder
Lambda (λ) Phage DNA
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
Genomic DNA, regardless of the source, can be digested with restriction enzymes that recognize….
4-8 consecutive bases
As these recognition sites occur in the DNA, the enzyme will cut the DNA at the restriction enzyme site
Restriction Enzymes
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.
Gel Electrophoresis
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.
Agarose
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%
Agarose Gel Electrophoresis
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.
How do we see the DNA?
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!
Consensus sequence
The DNA sequence that the restriction enzyme finds, binds to, and “cuts” the DNA.
The consensus sequence for HindIII is AAGCTT.
Why do we need a TBE running buffer?
Ensures the electric current flows through the gel
Maintains a stable pH, which is essential for proper DNA migration and separation
Linear DNA
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
Isoschizomers
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’
pBR322
A plasmid that contains many different RE sites and is used commonly in molecular cloning studies.
Also referred to as a cloning vector
Molecular cloning
The process of creating new DNA molecules for research and/or medical purposes
Restriction enzymes are often an important step to this process
When a restriction enzyme cuts the DNA and leaves sticky ends…
There are 5’ or 3’ DNA overhangs that are complementary
If sticky ends are complementary, then DNA molecules can be attached with greater ease.
When restriction enzymes cut the DNA leaving blunt ends…
There are no DNA overhangs
It is possible to attach (ligate) DNA molecules with blunt ends, but the efficiency is greatly reduced
Gel extractions
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.
In Plasmid Isolations, we want plasmid DNA from bacteria. What other components are in the bacteria that we do not want to isolate?
CRMP:
Chromosomal DNA
RNA
Membranes
Proteins
Resuspension Buffer (R)
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.
Lysis Buffer (L)
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.
Precipitation Salts (Psalts)
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
Silica columns/Column clean up
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)
Plasmid Isolation steps:
- isolate plasmids from bacteria
- digest (cut)
- ligation to create new plasmids
Alkaline Lysis Steps:
- Plasmid Isolation
Pellet bacterial culture and add resuspension buffer to prevent DNA degradation. Resuspension buffer contains RNAse so all RNAs will be degraded. - 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.
- 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).
The goal of plasmid isolations (or mini-preps) is to….
isolate and purify plasmid DNA from bacteria
Column Steps: Purification
- Binding buffer (added so the plasmid DNA can adhere to the silica membrane on the columns)
- 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.
Ampicillin Resistance
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
Amp vs Kan
Ampicillin INHIBITS new cell wall synthesis; satellite colonies
Kanamycin INHIBITS protein synthesis
Why do you add loading dye to each sample? How much and why that amount?
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
Factors that affect DNA mobility through an agarose gel:
% of agarose
Time
Size fragments of DNA (larger fragments stay up top)
Electricity (higher voltage → faster movement)
Shape (linear DNA vs plasmid)
What conditions can you change in the gel to change how fast (or slow) the nucleic acid moves through the gel?
Current
Concentration of the gel
How do you resolve (more clearly see) two bands that are close in size?
Time: Letting things run longer; more time to separate
Increase concentration of gel (ex. 1% to 2%)
Describe the mechanisms by which restriction enzymes work
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
“Ingredients” to set up a restriction digest
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
Name reasons why a restriction digest might not work.
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
How to figure out what type of enzyme you would use to excise a gene fragment from a plasmid
- Identify the Gene’s Location in the Plasmid
- Select enzymes that flank (cut before and after) the gene without cutting inside it.
- Choose an enzyme that works efficiently in standard buffer conditions.
How do you perform a gel extraction?
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
What is the end product of the alkaline lysis procedure (mini preps)?
a plasmid
Resuspenion buffer
resuspends pellet (initial pellet is bacterial cells, then we try to isolate plasmid from the cells)
lysis buffer
lyses things open
Precipitation salts
help to precipitate out the macromolecules (DNA, plasmid membrane, proteins) but leaves plasmids behind
Why do you not get chromosomal DNA from an alkaline lysis procedure?
Its big and doesn’t come back together correctly (fried egg is still protein but clear stuff is opaque)
Why do you not get proteins from an alkaline lysis procedure?
It doesn’t stain solution, psalts precipitate it out
Why do you not get RNA from an alkaline lysis procedure?
We use RNase to get rid of it
Why do you not get membranes from an alkaline lysis procedure?
It’s a macromolecule (phospholipid bilayer, proteins, and carbs) that gets precipitated out by psalts
What is the purpose of adding Magnesium and Calcium Chloride to the bacterial cells during this procedure?
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
Why do we perform a heat shock?
To make the membrane porous; allows plasmid to enter
We did a heat shock for 2min then put it back on ice
Transformation not efficient
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
**Why do we grow the bacteria for an hour after the heat shock?
[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
What are microsatellite colonies and why do they form?
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.
How do you know if the bacteria took up the plasmid?
Able to grow on selected media
Ones that took up amp grow on amp
Ones that took up kanamycin grow on kan
Cells that take up (transform) pBlu turn what color? Why?
Blue; the plasmid contains the lacZ gene, which encodes β-galactosidase
