L14 - Classical gene cloning 1 Flashcards
(11 cards)
define molecular cloning
the ways and reasons for copying pieces of DNA in a living cell
whats PCR cloning
amplifies fragment of DNA that can be inserted into host cell
- amplify sequene using PCR
- ligate PCR amplified DNA into a cloning ‘vector’
= this can be inserted into cell to ‘transform it’
describe the proscess and what shotgun cloning is
when you want to find/work out the DNA sequence for a particular protein or phenotype
- cut up genome of interest/target organism
- place all the different fragemnts into cloning vectors
- place each vector into a cell to create ‘genomic library’
= huge population of cells each containing a different piece of the original organism’s genome
- this library can now be ‘screened’ to find which cell with a particular fragment from orginal genome codes for our protein of interest
- final step is to amplify the vector and work out the target sequence
describe what a genomic library is
huge population of cells –> each containing a different piece of the original organism’s genome
we can screen for different fragments that code for different phenotypes and proteins as we please
= used for lots of different experiments
what is sub cloning
when you already have the genbe of interest but want to study it more
= move into a different vector
different vectors have different roles
expression vector, shuttle vector (movement between hosts), Broad host range vector ( ncan go into most bacterial species
name a commonly used plasmid cloning vecrtor and aome charecteristics of it
pUC18/19
A natural small and easy to work with plasmid that has been cut to remove non-required parts
can only replicate insde E.coli due to specific to this machinery
immobile –> ability of it to move is removed = prevent unwanted spread of potentially dangerous genes
single replication of origin and a dominant slectible marker
name a selectible marker that cpuld be used in gene cloning
B-lactamase
= breaks down penicillin/antibiotic = allows screening for which cells contain the desired plasmid
what are multiple cloning sites - MCS - and how are they used in screening
contain multiple different sites for different restruiction enzymes to cut and insert different desired DNA sequences
MCS are usally within reporter genes such as LacZ for blue/white screening
= if the gene we want is succesfully inserted into the MCS on the plasmid the reporter gene is disrupted
= so if the reporter gene is not expressing = we know succesfull insertion has occored
describe blue/white selection
XGAL is added
dark blue colour is shown due to beta-galctosidase breaking this down releasing bromochloroindol
if insertion has happened –> LacZ is disrupted and beta-galctosiadase is not produced
= white is shown sinstead as no breaking down of XGAL occors
why can we not use native E.coli for blue/white screening
normal E.coli produce Beta-galctosidase in their genome
= even if LacZ is disrupted on plasmid blue colour is still shown
what must we do to the E.coli for blue/white screening
alpha complementation
= disrupt the LacZ alpha subunit in E.colis Genomic DNA
means the only Beta-galctosidase that would be present is from the plasmid
