CLONING AND SUBCLONING DNA

Question 1

2 objectives of molecular cloning

Answer 1

-mutagenesis
-study expression of genes

Question 2

steps of molecular cloning (6)

Answer 2

-DNA preparation
-vector preparation
-union of DNA + vector by ligation
-incorporation of rDNA in recombinant cell
-cell division
-selection

Question 3

steps DNA preparation (4)

Answer 3

-cell lysis
-elimination of others molecules
-precipitation and concentration
-DNA selection with restriction enzymes

Question 4

a vector is a… with 1 nucteotide… overhanging on each sides

Answer 4

-plasmid
-T

Question 5

what enzymes do we need to insert the DNA in the TA vector (2)

Answer 5

ligase T4, alkanine phosphatase

Question 6

(5) methods to insert the rDNA

Answer 6

-physico chemical
-liposomes
-electroporation
-micro injection
-infection/transduction

Question 7

physico chemical method

Answer 7

-CaCl2 + thermic shock
-AND cation
-Polymers (PEG)

Question 8

3 methods of selection of recombinant clones

Answer 8

-hybridization
-immunochemistry
-phenotypic

Question 9

hybridization

Answer 9

labelled probe detecting cloned DNA/RNA transcript

Question 10

immunochemistry

Answer 10

encoded protein + specific antibody

Question 11

what are genomic DNA libraries coming from ?

Answer 11

genomic DNA

Question 12

what are cDNA libraries coming from ?

Answer 12

localization of clones