in vivo cloning

Question 1

What is the first enzyme?

Answer 1

= restricton endonuclease is used to cut out the gene, does not cut the gene cuts around it, to form a plarndromic base sequence- dna fragment intrested in
= enzymes cut at specific restriction sites- producing sticky ends
= modified to ensure transcription can occur

Question 2

What modifcation do we do?

Answer 2

= a promoter and a termnator region is added to the DNA ragment to allw transcriptiom

Question 3

What dt do we insert the NDA into?

Answer 3

= a vector
= carries isolated DNA rfagment
= into host cell
= commonly used as plasmids, crcular loop of DNA

Question 4

How do we insert the DNA into a vector?

Answer 4

= plasmid is cut open using the same restriction endonuclease enzyme- creates the same sticky end
= exposed nucleitdes on the dna fragment can form complembtary sticky ends on the plasmid

Question 5

How is the dna fragment and the plasmid stuck together?

Answer 5

= DNA ligase (glue)
= stixks them together the dna fragment and thr plasmid when combbinding
= catalyses the condensation reaction between the nucleoides, forming phosphodiester bondsn

Question 6

How is the vector inserted into the host cells?

Answer 6

= gene will be expressed
= cell emmbrane of the host cell must be more permeabke
= to increase the permeability host cells are mixed iwth Ca2+ ions and heat shocked enables thevector to enter host cells cytoplasm