Week2

What are the 5 steps of gene cloning?

Obtain gene of interest, insert into vector, transform host cell, recover cloned DNA for QA, express cloned DNA

What does DNA Ligase do?

creates binds on the 5’ phosphate and the 3’ hydroxy group to bind DNA strands together

What is another name for a restriction enzyme

endonuclease

Why do we need to perform a Q.A after DNA recovery?

to generate a stock

to determine if the DNA sequence is correct

What is the purpose of a “miniprep”

To isolate and purify DNA

What is ampR?

A ampicillin resistance gene casette

What is ori?

the origin of replication

What is TcR?

Tetracycline resistance gene cassette?

What is a gene casette?

A gene + the compatible DNA ends

What is another name for sticky ends?

cohesive ends

What does an MCS stand for?

Multiple cloning site

What is the purpose of inserting an MCS into a vector?

Act as an easy sequence for inserting new genes

What is the key functional feature of an MCS

They contain several restriction sites of known sequences

What is the LacZ gene?

A gene inserted which, when expressed with beta-galactosidase, will turn transformed bacteria blue

What method of vector purification is in minipreps typically

Alkaline lysis

How does alkaline lysis help to purify and extract plasmids?

It exposes the DNA into a solution

What are the steps of an alkaline lysis miniprep?

centrifusion, lysoyme, NaOH, KOAc

What chemical is KOAc

Potassium acetate

What enzyme is in minipreps?

lysozymes

What must be done as a secondary Q.A once DNA is extracted?

A260/A280 purity determination

How do restriction enzymes recognize where to cut

palindromic recognition sites

What enzyme will prevent EcoR1 from cutting at its restriction site?

EcoR1 methylase

What will travel further in electrophoresis, nicked DNA or linear DNA?

nicked DNA

What is nicked DNA

DNA with one bond broken in it

What is BioLabs

Where enzymes are purchased

How do we determine the chance of a recognition site occurring in a genome?

genome total bp x (1/4^n) where n = bp length of palandrome

What DNA ligase is typically used and what energy molecule does it utilize?

T4 DNA Ligase uses ATP

Where does a T4 DNA Ligase come from?

T4 Bacteriophage

What are the two molecules which can be used for energy by DNA Ligases depending on the specific ligase?

ATP or NAD+

What is the molar mass of a histone octamer?

108kD

What is the average molar mass of a nucleotide

330g/mol

What is this formula and what do N, P and f stand for?

% confidence formula

N= number of fragments required

P = percentage confidence

f = the fraction of the genome in each fragment

How many bp are wrapped around each histone?

200

How many base pairs are wrapped around a histone after trimming?

165bp

What is the size of thin chromatin fibers?

10nm

What is the size of thick chromatin fibers?

30nm

What enzyme trims histones?

MNase

What ions are required for PCR?

Mg

What is the purpose of Mg ions in PCR?

How would you describe Taq and Pfu DNA Polymerases?

Thermostable

Where do the primers bind?

To both the 5’ and the 3’ end of the melted strands

What is a dNTP

deoxynucleotide triphosphate (free nucleotide)

Which thermostable DNA Polymerase is proofreading?

Pfu

Which thermostable DNA Polymerase isn’t proofreading?

Taq

How many cycles are peformed in PCR

30

What are the three cycles of PCR?

denaturation, annealing and extension

What temperature does denaturation occur?

over 90

What temperature does annealing occur

50

What temperature does extension occur?

72

How long is a primer typically and what GC content does it have?

17-30bp, at least 40% GC content

Why is being 17-30bp and over 40% GC content important for a primer?

prevents primers from binding to themselves (primer dimers) and the formation of hairpin loops (too short for self binding)

creates solid annealing

How is a primer Tm calculated?

What end of the DNA strand do the primers bind to?

3’ end

What does it mean for a primer in PCR to be either forward or reverse?

Why is PCR not perfectly exponential growth?

renaturation of DNA can occur preventing replication

Why must the Tm of the two primers be within 2 degrees of each other

What happens if the primer Tm is exceeded

What happens if the PCR temperature is too far under the primer Tm

What is the pGEM-T System

A commersaily avalible open vector which is complementayr to the addittional nucleotide added by Taq

in the FASTA format, which strand is the coding sequence?

The top strand

What is a 5’ 3’ exonuclease activity?

proofreading

What secondary activity does Taq Polymerase have

a terminal transferase added on the 3’ end

(additional nucleotide requiring no template)

What is the prefered addittional nucleotide to be added by Taq and why?

A, prevent self bindign

What is the name of the additional nucleotide added by Taq

terminal transferase

What are common problems with PCR

digestion efficiency, ligation efficiency, avoiding recirculation of open vector

How do you avoid recirclisation of a cut vector?

Use two restriction enzymes with non-compatible ends

What does a phosphatase do?

How does a phosphatase prevent a vector from recirculating

What do you need to ensure is removed before electroporation

What is electroporation?

What is chemical transformation

How is chemical transformation acheived

What does the calculation for transformation efficency measure?

CFU/micro gram of DNA

What is the coefficient for pico

What does bacterial competency refer to?

Ability to transform

What is a transformant?

What does it mean to study something “in vitro”

study it outside of the organisation

Why do we use restriction enzymes on the entire genome when creating a vector?

To create fragments small enough to be able to be inserted into a plasmid

What is the concept of a gene library?

When a genome is spread across multiple vectors, a gene library is created

What is commonly done with a gene libirary once it has been created?

The entire library is transformed into a capable bacterium and it contains the entire genome

Why do all plasmids require an ORI

So that the plasmid containing the target sequence can be self-replicating within the bacteria it is inserted within

What does it mean for a bacterium to be naïve?

no previous exposure to [ whatever in context ]

If using genomic DNA for insertion into vector, how is the target sequence isolated?

amplification of only the target gene by designing exclusive PCR primers

If you needed to generate DNA from a sequence where only RNA is available, how would you achieve it?

Reverse transcriptase enzyme and PCR amplification

How do we know if the bacteria has transformed with the intended DNA?

lyse the bacteria, extract the DNA and perform a sequence analysis

Why must we do a QA when producing proteins from a vector?

To ensure the expression system of the organism is creating the intended protein

Once we are sure that the sequence transformed into the bacteria is correct through sequence analysis, what is performed?

expression of the gene in the form required to be studied

What part of a phage can be used as a vector?

a phage chromosome

Do plasmids have 5’ and 3’ ends?

Not unless they are cut

How is a coding sequence represented on a plasmid diagram?

an arrow

What is a phagemid

a hybrid of a phage and a plasmid

What is refered to by ori specificity

an ori recognised by the machinery of the specific organisim it is inserted within

What is crucial when selecting a restriction enzyme?

The restriction site doesn’t appear in the target sequence

Why would you use a phagemid

To utilize phage cell machinery for the expression of the target gene

What are the two chemicals which alkaline lysis relies?

NaOH (sodium hydroxide)

KOAc (potassium acetate)

What are the 4 ingredients for alkaline lysis?

Bacterial pellet

NaOH (sodium hydroxide)
KOAc (potassium acetate)

Lysozyme

What are the two options of chemical “washes” used after alkaline lysis?

Phenol chloroform

Cesium chloride + ethidium bromide

What are the benefits and risks of using cesium chloride + ethidium bromide over phenol chloroform?

It is expensive and toxic

But can help differentiate between types of DNA

Where do you extract the DNA from in the tube?

The very bottom as that is where the plasmids are located

Protein or phenol contamination is indicated by what A230/A260 ratio

over 0.5