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What is the general point of transformation?
It allows DNA to enter the inside of a bacteria cell from outside
What are the zones called that result from the cell growing and dividing quickly and the membrane not being complete
What kind of charge of DNA have?
What causes this charge?
- The phosphate groups
What ion is used to balance the charge of DNA during a transformation reaction?
Why do you cool your cells during a transformation?
To slow the movement of the membrane
Why do you heat shock your cells during a transformation?
It helps sweep the DNA into the cell
What is E. Coli's general purpose in molecular biology?
To propagate DNA
How often does E. Coli divide?
About every 20-30 minutes
during what phase do we do transformations?
during he lag phase
Ampicillin is what kind of antibiotic? (bacteriostat/bacteriocide)
Explain the mechanism through which Ampicillin works
- Has a beta-lactum ring structure, which binds to transpeptidase, rendering it inactive. (transpeptidase usually pulls cell wall together) The cells lyse because they take on too much material from outside the cell.
Kanamycin--Bacteriostat or bacteriocide?
How does it work?
- Binds to the ribosome, and the cells can't produce proteins anymore. They die when trying to divide
When your DNA has been added to the bacterial cell, it joins with the DNA previously in there to form one extra long plasmid, True or False?
False, you have the plasmid you added plus the original bacterial DNA, but they remain separate from each other
What is the mechanism of being Ampicillin-resistant?
Cells that are ampicillin resistant are able to break the beta lactum ring
What does GFP stand for and where does it come from?
- Green Fluourescent Protein
- From Jellyfish
Through what reaction is kanamycin resistance facilitated?
The kanamycin is phosphorylated with a phosphate group added in place of a hydroxyl group
Does E. Coli naturally have a low or high ability to take up DNA from the environment?
Who proposed the idea of restriction enzymes?
Luria, Bertani, and Weigel in 1952-1954
Why are restriction enzymes named as such?
Becuase the researchers just noticed that something was "restricting" the growth of the virus, thus coining the name restriction enzyme
Who proposed the mechanism and modification of lambda phage DNA after infection?
Who discovered the first type 2 restriction enzyme?
Who is known for genome mapping?
How many types of restriction enzymes are there?
Explain how type 1 restriction enzymes work, as well as what they need to function
- cut at a certain distance away
- have 3 subunits, sites are assymetrical
- require ATP, Mg2+
Explain how type 2 restriction enzymes work and what they need to function
- sequence-specific endonucleases
- recognize and cut within pallindromic DNA sequences
- Require Mg2+
Explain type 3 restriction enzymes
- recognize 2 separate sequences
- require ATP
- contain mulitple subunits
what type of reaction do restriction enzymes perform?
hydrolysis reaction--cleaves the DNA by breaking the phosphodiester bond, adds water between the bond
Transformation efficiency = ?
- colony forming units/ ug DNA
- (micrograms DNA)
Which type of restriction enzymes did we generally use in class?
What does the restriction enzyme buffer used in lab consist of, and what does each chemical do?
- TrisCl--maintains appropriate pH
- Mg--necessary cofactor of restriction enzyme
- Salt--maintains isotonic environment
at what temperature do restriction enzymes function optimally at?
What three scientists were awarded the Nobel Prize in 1978 for their work in "restriction enzymes and their application to problems of molecular genetics"
Arber, Smith, and Nathans
Where does lambda DNA come from?
it is purified from bacteriophage lambda
About how many basepairs is Lambda DNA?
Is lambda DNA linear or circular?
- Trick question :)
- Usually linear, but can be circular
What is included in TAE buffer and what does each component do?
- Acetate--provides ions
- EDTA--is a chelator, binds to Mg2+ ions
What is the function of Ethidium Bromide?
- It makes the DNA bands visible under the UV light
- It intercalates between the rungs of the DNA
What is the function of loading dye?
- increases the density of the sample
- allows us to see where the DNA is so we don't run it off the gel
What two materials are in loading dye?
- bromophenol blue--the lower color
- xylene cyanol--the upper color
Give the general outline of Plasmid Isolation
- isolate and grow antibiotic resistant cells
- harvest cells using differential centrifigation
- lyse the bacteria
- precipitate cell debris and genomic DNA
- precipitate DNA and RNA
What are the 3 components in GTE buffer and what are their functions?
- Glucose--maintains osmolarity
- Tris--buffers pH
- EDTA--binds divalent ions, weakening the cell envelope, also protects the DNA
What does SDS stand for and what is its function?
- Sodium dodecyle sulphate
- it acts as a detergent and lyses the cell and unfolds/denatures proteins
What is NaOH and what is its function in a transformation reaction?
- Sodium hydroxide
- it's a strong base, which deprotonates the nucleotides, causing the DNA to become single stranded
What does KoAc stand for?
What makes it up?
What does it do?
- Potassium Acetate/Glacial Acetic Acid
- Potassium Acetate: anything associated with SDS forms a precipitate with this and comes out of solution
- Glacial acetic acid: brings the pH back down, allows the DNA to renature
- Plasmids go back together well, but more complex DNA doesn't
What use does ethanol have in a transformation reaction?
It is used to dehydrate the DNA. When added, the DNA precipitates out of solution
Why is RNAase added to a tranformation reaction?
It chews up the remaining RNA, leaving us with only DNA in tact
Ligation is what kind of chemical reaction?
What does it do?
What does it require?
- reseals the DNA
- requires ATP and appropriate sticky ends
Buffer solution contains all of this
Who is credited with the process of PCR amplification?
What year did they win their Nobel Prize?
- Carey Mullness, came up with PCR in mid 80's
- Nobel Prize in 93
What are the 3 steps in a PCR reaction?
What temperatures do they happen at?
- 1) Denaturing, 95C--denatures DNA into single stranded DNA
- 2) Anneal Primers, 50-68C, from hydrogen bonds (hydrolyze)
- 3) Primer Extension/Synthesis, 72C--makes more copies of the DNA you want
About how long is each step of the PCR reaction?
How many bp/sec are added during step 3 of PCR?
What are the requirement for PCR?
- template (source DNA)
- Taq polymerase
- Buffer containing MgCl2, which helps with the specificity of the reaction
Where does taq polymerase come from?
About how long are primers used in PCR reactions usually?
Are primers single stranded or double stranded?
Why is having a GC content of approximately 50% important of a primer used in PCR?
There are 3 hydrogen bonds between G and C, and ensuring that the primer has a 50% GC ratio ensures a more stable molecule to work with
Enables an increase in temperature, which increases the specificity of the annealing
Two kinds of primers are required for each PCR reaction. What are their names and what does that mean?
Which way must they be oriented?
Upstream primer--5' primer, used as a template for the bottom strand of DNA
Downstream primer--3' primer, used as a template for the upper strand of DNA
The 3' ends must be facing each other
Is the primer discarded after the PCR reaction is finished?
No, it becomes part of the DNA
What does taq polymerase do?
It adds nucleotides to the primer that has already bound to the DNA
what does PTC stand for?
what does PCR stand for?
Polymerase Chain Reaction
What is the ability to tast PTC due to? (On the DNA level)
It is due to a single nucleotide polymorphism (SNP), which is a change in a single nucleotide
Taq polymerase has something funny that it does...what is that?
It has a 3' terminal transferase activity, which adds an A at the end of the sequence
When using blue/white screening, what chemical are we using?
Which colonies have the insert?
- We are using xgal
- the white colonies have the insert
about how manhy bp is the PTC receptor fragment?
When multiple plasmids are linked together, what is this called?
Where will you find them on a gel?
- They will not move very far, if at all
To what end does taq polymerase add nucleotides?
the 3' end
Describe the difference between transformation and transfection
- Transformation inserts genetic material into bacteria
- Transfection inserts genetic material into a mammalian cell
Why do we use agarose gels and polyacrylamide gels?
Which kind of gel forms smaller pores?
- Agarose gels allow us to separate chunks of DNA/plasmids
- Polyacrylamide gels allow us to separate proteins
- Polyacrylamide has smaller pores
Is polyacrylamide toxic to humans?
Yes, it is a cumulative neurotoxin
What two part are necessary in a polyacrylamide gel? What are their purposes?
Stacking (upper) gel--gets the proteins all lined up to go through the smaller pores, has a lower concentration of polyacrylamide
Resolving (lower) gel--actually separates out the proteins, has a higher concentration of polyacrylamide
Why do you have to use 2 different buffers in a polyacrylamide gel?
What is this called?
- Because the two gels have different pH's
- Called a discontinuous buffer system
Approximately what are the pH's of the two gels? (in SDS Page)
- resolving gel: 8.8
- Stacking gel: 6.8
What chemicals are present in the buffer used in SDS PAGE?
What do they do?
- Tris--stabilized the pH
- Glycine--runs behind your protiens of interest, kind of pushes them through
- SDS--binds to the linear molecule and makes it negatively charged
What is in the protein sample buffer used in SDS page?
This is what the samples are prepared in before running
- 2-mercaptoethanol/beta-mercaptoethanol--prevents oxidation of cystines and breaks disulphide bonds
- SDS--detergent, adds negative charge to the proteins
- Glycerol--weighs down the sample
Describe what we mean by sandwich, and what it contains
- bubble paper
- bubble paper
What kind of membrane do we use in the SDS page procedure?
What chemical is important to maintain the shape of the gel?
What else does methanol do?
As the proteins come out, they are covered with SDS, and are negatively charged. Methanol pulls off the soad, and sticks to the membrane
How many antibodies do you need when doing immunoblotting?
Explain the function of the different antibodies used
Primary antibody--is against the protein of interest and binds to it (you can't see it)
Secondary antibody--used to detect the primary antibody
What does the secondary antibody bind to during immunoblotting?
YFP--> alkaline phosphatase (still can't see them), phosphate group is dephosphorylated, forms a blue compound.
What does NBT act as?
- An oxidizer, so it gets reduced. It is a deep blue color
- This is the blue band that we see