Cloning Practical 1

Question 1

What is the first step of cloning

Answer 1

Bioinformatics search to sequence wanted gene (to design PCR primers)

Question 2

Which enzyme is used to cut recombinant plasmid pblt101 after using it to cut required dna

Answer 2

Ecor1 (restriction endonuclease)

Question 3

Where does ecor1 cut

Answer 3

Gaattc palindromic sequence causing sticky ends

Question 4

How are sticky ends from ecor1 in the pblt101 plasmid annealed

Answer 4

Dna ligase reforms phosphodiester bonds

Question 5

Where is the transporter protein inserted in pblt101 plasmid

Answer 5

At MCS of the plasmid between the lacz gene

Question 6

What is the MCS

Answer 6

Multiple cloning site is an area where restriction endonuclease recognise on plasmid dna

Question 7

What are cells treated with for transformation with both pblt101 and pic19h

Answer 7

Cacl

Question 8

Why is cacl iced important in transformation

Answer 8

Ca allows dna to be close to lps on the ecoli membrane (both negative)

Question 9

Why are cells put back on ice after heat shock

Answer 9

Recover cell wall

Question 10

Why was ecoli JM05 used

Answer 10

Lack of endonuclease activity to keep dna intact