Exp 3: Pre-prac - cloning interactive

Question 1

What are the 5 main stages involved in the cloning process? (in order)

Answer 1

Restriction Digest
Ligation
Transformation
Selection
Check for Insert

Question 2

Name a way of removing the unwanted donor plasmid DNA

Answer 2

gel electrophoresis

Question 3

What are the end products of the restriction digest in cloning?

Answer 3

Insert
Unwanted donor plasmid DNA
Linearized recipient DNA

Question 4

What process describes combining the insert and recipient plasmid? And what enzyme is used?

Answer 4

ligation
via DNA ligase

Question 5

What is the name given to the product after ligation of a recipient plasmid and insert?

Answer 5

recombinant plasmid

Question 6

What is a “competent” cell?

Answer 6

A cell with a changed cell wall that allows bacteria to take up the plasmid DNA

Question 7

What process might be used to create competent cells?

Answer 7

heat or electricity

Question 8

What is transformation in cloning?

Answer 8

The process of making competent cells and stimulating them to make more copies of our plasmid

Question 9

What is the name given to the process of removing cells with no plasmid from those with and replicating those with?

Answer 9

Selection

Question 10

How is selection achieved in cloning bacterial cells? Which cells survive?

Answer 10

Plasmids contain a selection marker (usually antibiotic resistance)
bacteria onto agar with antibiotic
Cells with the antibiotic resistance gene survive i.e. those with that have takne up hthe plasmid

Question 11

After selection of cells (during cloning) using agar with an antibiotic on has taken place, can we be sure that the cells which have survived have taken up our plasmid with the desired insert? Why/why not?

Answer 11

No
Because the bacterial cells that have survived could have taken up a different plasmid which is also resistant to the antibiotic (i.e. contains the antibiotic resistance gene)
e.g. the recipient plasmid could have recombined with itself and not the insert

Question 12

What processes would we used to check for an insert in a plasmid taken up by a bacterial cell?

Answer 12

Purify the DNA
Digest with the original enzyme (restriction nuclease)
Analyse the digested fragments using gel electrophoresis

Question 13

When checking an insert has been incorporated into a plasmid (for cloning) the purified DNA undergoes a digest with the original restriction nuclease, what would be characteristic of gel electrophoresis if the insert is present vs not present?

Answer 13

If the insert is present there will be an extra band in the gel electrophoresis (insert band and main plasmid/cloning band