Experiments

Question 1

Describe the series of steps to isolate genomic DNA from an E. coli culture

Answer 1

Principles and key steps of DNA/RNA isolation/purification:

• Step 1. Breaking open the cells to release DNA/RNA
• Step 2. Separate DNA/RNA from protein

Traditional Phenol extraction

Column-based extraction: Silica and anion exchange resins

• Step 3. DNA purification

Removal of RNA using ribonuclease

DNA purification via ethanol precipitation

Question 2

Step 1. Breaking open the cells to release DNA/RNA

What reagents can be used?

Answer 2

Lysozyme→To digest the peptidoglycan layer of the cell wall

Detergent→ To dissolve the lipids in the cytoplasmic membrane

EDTA(ethylene diamine tetraacetate)→ To chelate or remove the metal ions that bind components of the outer membrane together in gram-negative bacteria

Question 3

Name methods to:

Step 2. Separate DNA/RNA from protein

Answer 3

• Traditional Phenol extraction→Phenol, also known as carbolic acid, very corrosive and extremely dangerous, because it dissolves and denatures proteins.
• Column-based extraction: Silica and anion exchange resins

Question 4

Describe Step 3 DNA purification

Answer 4

• Removal of RNA using ribonuclease
• DNA purification via ethanol precipitation

Question 5

Describe Step 4. Determination of the quality and quantity of the purified DNA

Answer 5

• Spectrophotometry- ultraviolet (UV) absorbance measurement
• Agarose Gel Electrophoresis

Question 6

Explain the Principles of agarose gel separation

Answer 6

• DNA is negatively charged and migrates through the gel to the positive electrode.
• Agarose separates DNA fragments on the basis of size.

Question 7

How do we determine the quality of the purified DNA?

Answer 7

Agarose Gel Electrophoresis

From the image:

(A) Trans2K™ Plus DNA Marker

(B) acceptable/suitable sample

(C) degraded sample

(D) sample contaminated with RNA

(E) sample contaminated with protein. Red boxes denote areas of contamination.

Question 8

Strategies to obtain artificial DNA: PCR

Answer 8

•Polymerase Chain Reaction (PCR):

a laboratory technique for DNA replication that allows a “target” DNA sequence to be selectively amplified. PCR can use the smallest sample of the DNA to be cloned and amplify it to millions of copies in a short period of time.

Question 9

Strategies to obtain artificial DNA: RT-PCR

Answer 9

Reverse transcription PCR (RT-PCR):

A laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using PCR.

Question 10

What are the advantages of using mRNA?

Answer 10

• Represents only contiguous coding material - introns occupy 9/10ths of a eukaryote gene. Hence using mRNA can get a gene into a vector
• mRNA free from wild type promoters
• mRNA represents the genes most actively coding for proteins - only 2% of genomic material is involved in protein coding