In Vivo Gene Cloning Flashcards
What are the 2 antibiotic resistance genes in R plasmids?
Ampicillin and Tetracycline
Where does the restriction endonuclease cleave the R plasmid?
In the Tetracycline resistance gene
What are the three types of gene markers?
- enzyme markers
- antibiotic resistance
- fluorescent markers
What is replica plating?
using wood and velvet to transfer colonies of bacteria onto a new agar plate in the same location
What happens to the replica cultures that contain DNA fragments when using antibiotic resistance as gene markers?
they will die
Where does the restriction endonuclease cut plasmids containing gene markers?
through the gene (FGP, Tetracycline, Lactase)
What is the colour change caused by lactase?
colourless to blue
What might prevent a bacterial host from taking up the vector containing the DNA?
- plasmids rejoin, so no DNA even if taken up
- DNA fragments join together
- transformation doesnt occur
How is transformation of the vector into the host done?
Ca2+ ions, on ice
temperature increase
make the membranes more permeable so plasmids can enter
Not always successful
How are DNA fragments prepared?
promoter and terminator sections added
cut with same restriction endonuclease
Why are promotor regions necessary?
so RNA polymerase and transcription markers can bind and allow protein synthesis
Why are terminator regions necessary?
to stop protein synthesis
Why might a gene be inserted into egg cells rather than organisms?
-as the gene would be present in most cells
- so would get into the cells that make desired protein
Why are gene markers used?
not all cells would have taken up plasmid
not all plasmids would have taken up DNA fragment
organisms/cells that have successfully taken up plasmid and DNA will be identified (glow etc.)
Why is it important that genes are expressed only in the correct cells?
-so protein can be harvested
- so protein will not cause harm in other cells
