Recombinant DNA Techniques
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29 Terms
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 restriction sites
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+