vectors & libraries

Question 1

libraries defintion

Answer 1

random collection of DNA

Question 2

genomic libraries contain…

Answer 2

all DNA

Question 3

cDNA libraries contain…

Answer 3

exons, coding genes only

Question 4

libraries purpose

Answer 4

isolate DNA fragment to analyze

Question 5

vector purpose

Answer 5

amplify and analyze DNA

Question 6

what plasmid are we using

Answer 6

pTriplEx

Question 7

cDNA library steps

Answer 7

1) isolate mRNA
2) purify mRNA
3) reverse transcribe mRNA
4) remove RNA from RNA-DNA hybrids
5) perform second strand DNA synthesis
6) ligate double stranded cDNA into pTriplEx2 vector
7) transform to bacteria

Question 8

what gene is used for plasmid

Answer 8

lacZ gene

Question 9

what are the restriction sits of lacZ gene

Answer 9

SFiiA and SfiiB

Question 10

vector steps

Answer 10

1) digest - gene of interest is cut to fit vector (plasmid)
2) ligate - attach gene of interest into vector
3) transform - insert vector into host bacteria (bacteria will amplify it)

Question 11

what bacteria do we inject the gene of interest into

Answer 11

E.Coli

Question 12

how to know if insert is in plasmid

Answer 12

selectable markers

Question 13

how to know if plasmid is in bacteria

Answer 13

ampicilin resistance gene, lacZ gene

Question 14

what will happen if the bacteria has no plasmid

Answer 14

the bacteria won’t grow

Question 15

AmpR gene function

Answer 15

helps grow bacteria in tough conditions, antibiotic

Question 16

LacZ gene function

Answer 16

shows if plasmid contains insert, encodes beta-galactosidase enzyme

Question 17

what color will the colonies be if insert is in colonies

Answer 17

white (disrupts lacZ gene activity)

Question 18

what color will the colonies be if insert is not in colonies

Answer 18

blue

Question 19

central dogma

Answer 19

genetic info only flows in one direction - DNA, RNA, Protein