DNA Purification

Question 1

What is the first step in the DNA purification process?

Answer 1

Cell lysis.

Question 2

What anionic detergent is used for cell lysis?

Answer 2

Sodium dodecyl sulfate (SDS).

Question 3

What DNA purification method gives higher purity but uses more harmful chemicals?

Answer 3

Phenol/chloroform extraction.

Question 4

What is on the bottom layer - ‘organic phase’ - after phenol/chloroform centrifugation?

Answer 4

Lipids and phenol/chloroform.

Question 5

How many layers are formed after phenol/chloroform centrifugation?

Answer 5

3

Question 6

What solution is added in the protein precipitation method of purification?

Answer 6

Protein precipitation solution (PPS).

Question 7

After either methods of DNA purification, where is the DNA?

Answer 7

In the supernatant.

Question 8

Why does isopropanol precipitate DNA out of solution?

Answer 8

DNA is insoluble in alcohols.

Question 9

Following DNA purification, what is the next step?

Answer 9

DNA precipitation.

Question 10

What co-precipitant helps us see the DNA after isopropanol precipitation?

Answer 10

Glycogen.

Question 11

Ethanol precipitation involves ethanol and what other reagent?

Answer 11

Sodium acetate.

Question 12

How does sodium acetate reduce DNA’s solubility for precipitation?

Answer 12

Sodium cations neutralise the negative phosphates in DNA, thus lowering hydrophilicity.

Question 13

After DNA precipitation and before rehydration, what is done with the DNA pellet?

Answer 13

The DNA pellet is resuspended in 70% ethanol to wash any reagents away.

Question 14

What is the final step of the entire DNA purification process?

Answer 14

Rehydration.

Question 15

What A260/A280 values indicate purity?

Answer 15

1.8-2.0

Question 16

At what wavelength do proteins absorb UV light?

Answer 16

280 nm (A280)

Question 17

What would we use to find the A260/A280 ratio of a sample?

Answer 17

NanoDrop spectrophotometer

Question 18

When are the three stages that intercalating dyes can be added for gel electrophoresis?

Answer 18

1. In the molten gel, pre-casting.
2. In the buffer.
3. Post-electrophoresis.

Question 19

Why are they called intercalating dyes?

Answer 19

They intercalate the bases of the DNA ladder.

Question 20

Restriction enzymes are what kind of nuclease?

Answer 20

dsDNA endonuclease.

Question 21

The bases of RE recognition sequences must be what?

Answer 21

Palindromes.

Question 22

What does ‘N’ refer to in a recognition sequence?

Answer 22

Any nucleobase.