Insertional Activation

Question 1

What is blue-white screening?

Answer 1

is a method that allows a research to determine which transformants have a recombinant plasmid and which do not.

Question 2

What do white colonies contain?

Answer 2

they are the ones that have the recombinant plasmid a non functional lacz gene

Question 3

What do blue colonies contain?

Answer 3

they are the ones without the recombinant plasmid thus a functional lacz gene

Question 4

What gene is involved with blue white screening?

Answer 4

the lacZ gene on pUC18

Question 5

What does a recombinant plasmid contain?

Answer 5

DNA from two different sources

Question 6

What are the important components of a vector?

Answer 6

antibiotic resistance gene, ori, and polylinker

Question 7

What is pUC18?

Answer 7

It is the most commonly used plasmid in molecular biology

Question 8

What do most plasmids contain?

Answer 8

a antibiotic resistance gene

Question 9

What antibiotic resistance gene does pUC18 contain?

Answer 9

ampR

Question 10

What is the first purpose of the antibiotic in the media?

Answer 10

to get rid of the bacteria that did not take up the plasmid

Question 11

What is the second purpose of the antibiotic in the media?

Answer 11

to grow the bacteria in the plasmid in the media containing the antibiotic so they dont lose the plasmid.

Question 12

What is a polylinker?

Answer 12

it has all of the restriction enzyme sites

Question 13

What is the Ori?

Answer 13

origin of replication

Question 14

What is the first step in constructing a recombinant plasmid?

Answer 14

the DNA of interest and plasmid of interest are digested in the same restriction enzyme

Question 15

What is the second step in constructing a recombinant plasmid?

Answer 15

the two are ligated together needing a 4 to 1 ratio of insert to vector

Question 16

What is the third step in constructing a recombinant plasmid?

Answer 16

the ligation mix is transformed into E. coli and plated on the medium that has the antibiotic.

Question 17

What is the fourth step in constructing a recombinant plasmid?

Answer 17

the colonies are screened to determine which have plasmids with inserts

Question 18

What is insertional activation?

Answer 18

it is the interruption of a gene on a plasmid that occurs when an insert is ligated into the gene

Question 19

What does insertional inactivation allow?

Answer 19

it allows for the screening of colonies transformed with the ligation mix to determine which ones have the plasmid with the insert and which ones do not.

Question 20

What can the gene be in insertional activation?

Answer 20

it can either be a antibiotic resistance gene, but needing two of these. or a gene like lacZ that encode an enzyme that can change the colorless substrate called X gal into a product that is blue.

Question 21

What two types of colonies will appear when E. coli transformed with the ligation mix is plated on media with its corresponding antibiotic resistance gene?

Answer 21

1. those that have the vector that is closed up and does not contain an insert.
   2 those that have the vector that ligated to the insert

Question 22

How many antibiotic resistance genes does a vector need?

Answer 22

2 different antibiotic resistant genes

Question 23

what does lacZ encode?

Answer 23

beta galactosidase

Question 24

what does beta galactosidase normally do? what does it do now?

Answer 24

it breaks down lactose with colorless products glucose and galactose, it breaks down X gal now and turns it blue