Cloning

Question 1

Definition of nucloid?

Answer 1

Circular chromosomal DNA in prokaryotes.

Question 2

Which one gene do plasmids usually encode for?

Answer 2

antibiotic resistance gene

Question 3

What do bacteria use to make multiple copies of a plasmid, or pass it down to offspring?

(region where DNA replication begins in plasmid)

Answer 3

Origin of replication (Ori)

Question 4

Where in the plasmid do we insert our gene of interest?

Answer 4

Multiple cloning site (MCS)

Question 5

What are the steps of DNA cloning?

Answer 5

1. Cut DNA
2. Mix pieces together
3. Ligation
4. Transformation
5. Selection

Question 6

Which bacterial enzymes recognise palindromic sequences and are used to cut DNA?

Answer 6

Restriction endonuclease

Question 7

Which areas are complementary between the bacterial plasmid and the gene of interest?

Answer 7

Sticky ends

Question 8

After sticky ends bind, they are not covalently bound. How is this done?

Answer 8

Form DNA backbone using T4 DNA ligase.
Uses ATP to combine phosphate and hydroxyl, reforming phosphodiester bond.

Question 9

What are the two methods of DNA transformation in cloning?

Answer 9

1. Chemical transformation (heat shock)
2. Electroporation

Question 10

Why is it difficult for DNA to enter through the E.coli membrane?

Answer 10

Head groups of lipids on membrane are negatively charged.
DNA is also negatively charged.
Repel each other.

Question 11

What is added to the E.coli to make them chemically competent? And what does it do?

Answer 11

Calcium chloride.
Calcium chloride ions are positive.
They neutralise the charge of the polar head groups.

Question 12

What are the steps of chemical transformation (heat shock)?

Answer 12

• Addition of calcium chloride.
• Put competent cells on ice (to stabilise membrane).
• Move competent cells to 42 degrees (pressure difference so plasmid enters).
• Move cells to 39 degrees for recovery (so pores can heal).

Question 13

Why is transformation using heat shock called chemical transformation?

Answer 13

Because it involves mixing plasmid DNA with chemically competent cells.

Question 14

How does electroporation work?

Answer 14

Brief burst of electrical shock.
Pore formation in bacterial membrane.
Plasmids enter bacteria.

Question 15

One pro and one con of electroporation as a transformation technique?

Answer 15

Pro: higher transformation efficiency
Con: more complicated than heatshock method as need electroporation kit

Question 16

What is a mini prep?

Answer 16

Technique that isolates plasmid DNA from bacterial cells.

Question 17

What is a restriction digest?

Answer 17

Cutting of DNA by restriction enzymes at specific locations

Question 18

How can plasmids be verified using restriction digest?

Answer 18

Use same endonucleases as previous to cut DNA.
Run the digest on agarose gel.
Two bands should appear:
1. insert
2. vector