blue/white screening Flashcards
what does blue/white screening allow?
detection of recombinant bacteria
what is the competent host cell grown on?
X-gal
what is X-gal?
galactose linked to indole (it is a colourless analog of lactose)
cells transformed with vectors containing recombinant dna form what colour colonies?
white
how is a gene insterted into a plasmid vector?
ligation
what is a problem with transformation of desired genes?
not all plasmids that are transformed into cells may contain the desired gene insert
what do blue colonies show in regard to insert ?
may contain vector with uniterrupted lacZ a (therefore no insert) B GALACTOSIDASE IS FUNCTIONAL
what do white colonies show in regard to insert?
indicate presence of insert. Insert in lacZ a that is disrupting the formation of an active B galactosidase
why shouldn’t the media include glucose?
glucose affects lac operon (the protein involved in glucose important shits down lactose permease when glucose is present)
what must the plasmid contain?
lacA
what must the e.coli (competent cell) contain?
mutant lacZ gene with deleted sequences
what is X gal sensitive to?
light therefore should be stored in the dark.
what are some drawbacks of blue/white screening?
ligated dna may not be the correct one,may not have been properly ligated,mutation may lead to the alpha fragment not being expressed, finally a colony with no vector will also appear white .
why may blue colonies actually contain the insert (wider)?
If insert is “in frame” with lacZalpha gene and the STOP codon is absent in the insert. This can lead to expression of a fusion protein that has functional lacZa if it’s structure is not disrupted.
who discovered the principle of a-complementation of the b-galactosidase gene? (wider)
Agnes Ullman
