21.2 In vivo gene cloning - the use of vectors Flashcards
1
Q
what does in vivo mean?
A
inside living organisms
2
Q
main points for in vivo
A
- (transcription) DNA fragment prepared using promoter and terminator
- plasmid inserted into cell using transformation
- cells checked for plasmid
- cells checked for plasmid with DNA using marker genes
3
Q
What is a promoter?
A
base sequence that allows the binding of the RNA polymerase
4
Q
what is a terminator?
A
base sequence that stops the RNA polymerase
5
Q
How do you put the DNA fragment into a plasmid?
A
- cut the plasmid and DNA fragment with same RE
- produces sticky ends
- complementary base pairing
- DNA ligase joins nucleotides together
6
Q
How is transformation used to insert the plasmid into the cells?
A
add Ca2+
and change temperature
7
Q
How do you identify the uptake of the plasmid?
A
Plasmid with ampicillin resistance gene and tetracycline resistance gene
- grow in ampicillin
= those without gene will die
8
Q
main points for determining gene present?
A
- replica plating
- use marker gene
- those that don’t show the marker gene contain the DNA
- refer back to cloned colonies
9
Q
how do you do replicate plating?
A
- make copies of the colonies on agar plate
- use velvet on a wooden table
