Insertional Activation Flashcards
What is blue-white screening?
is a method that allows a research to determine which transformants have a recombinant plasmid and which do not.
What do white colonies contain?
they are the ones that have the recombinant plasmid a non functional lacz gene
What do blue colonies contain?
they are the ones without the recombinant plasmid thus a functional lacz gene
What gene is involved with blue white screening?
the lacZ gene on pUC18
What does a recombinant plasmid contain?
DNA from two different sources
What are the important components of a vector?
antibiotic resistance gene, ori, and polylinker
What is pUC18?
It is the most commonly used plasmid in molecular biology
What do most plasmids contain?
a antibiotic resistance gene
What antibiotic resistance gene does pUC18 contain?
ampR
What is the first purpose of the antibiotic in the media?
to get rid of the bacteria that did not take up the plasmid
What is the second purpose of the antibiotic in the media?
to grow the bacteria in the plasmid in the media containing the antibiotic so they dont lose the plasmid.
What is a polylinker?
it has all of the restriction enzyme sites
What is the Ori?
origin of replication
What is the first step in constructing a recombinant plasmid?
the DNA of interest and plasmid of interest are digested in the same restriction enzyme
What is the second step in constructing a recombinant plasmid?
the two are ligated together needing a 4 to 1 ratio of insert to vector
What is the third step in constructing a recombinant plasmid?
the ligation mix is transformed into E. coli and plated on the medium that has the antibiotic.
What is the fourth step in constructing a recombinant plasmid?
the colonies are screened to determine which have plasmids with inserts
What is insertional activation?
it is the interruption of a gene on a plasmid that occurs when an insert is ligated into the gene
What does insertional inactivation allow?
it allows for the screening of colonies transformed with the ligation mix to determine which ones have the plasmid with the insert and which ones do not.
What can the gene be in insertional activation?
it can either be a antibiotic resistance gene, but needing two of these. or a gene like lacZ that encode an enzyme that can change the colorless substrate called X gal into a product that is blue.
What two types of colonies will appear when E. coli transformed with the ligation mix is plated on media with its corresponding antibiotic resistance gene?
- those that have the vector that is closed up and does not contain an insert.
2 those that have the vector that ligated to the insert
How many antibiotic resistance genes does a vector need?
2 different antibiotic resistant genes
what does lacZ encode?
beta galactosidase
what does beta galactosidase normally do? what does it do now?
it breaks down lactose with colorless products glucose and galactose, it breaks down X gal now and turns it blue
