21.2 in vivo gene cloning Flashcards
what are recognition sites?
sequence of DNA that is cut by restriction endonucleases
why is the same restriction endonuclease used in the plasmid as the DNA?
fragments produced will have sticky ends that are complementary to one another
what does DNA ligase do?
joins DNA fragment with plasmid
what method do you have to use to prepare the DNA fragment for insertion?
- add promoter
2. add terminator
what is a promoter?
region of DNA that provides a binding site for RNA polymerase to attach
what is a terminator?
region of DNA the releases RNA polymerase
what is a vector?
carrier of DNA into a host cell
what is the most common vector?
plasmid
explain the method for inserting the DNA fragment into a vector
- plasmid and DNA cut using the same restriction endonucleases
- mix plasmids and DNA together
- add DNA ligase
what is transformation?
process by which plasmids are reintroduced to bacteria cells
explain the method for transformation
- mix plasmids and bacterial cells together in a medium containing Ca2+ ions
- heat
why will only 1% of the bacterial cells take up the plasmids?
- some plasmids will have closed up without incorporating the DNA fragment
- some DNA fragment ends join together to form its own plasmid
what are marker genes?
genes used to identify whether a gene has been taken up by bacterial cells
what are 3 types of marker genes?
- antibiotic resistant marker
- fluorescent marker
- enzyme marker
how do fluorescent markers work?
- if plasmid has been taken up bacteria doesn’t fluoresce
- if plasmid hasn’t been taken up then fluoresces
how do enzyme markers work?
- if plasmid has been taken up, bacteria doesn’t produce enzyme
- if plasmid hasn’t been taken up, bacteria produces enzyme
how do antibiotic- resistant markers work?
- if plasmid has been taken up, bacteria is resistant to 1 antibiotic
- if plasmid hasn’t been taken up, bacteria is resistant to 0 antibiotics
- if plasmid has been taken up but doesn’t have DNA fragment, bacteria is resistant to 2 antibiotics
