LSM1102

MOLECULAR GENETICS Laboratory Sessions

Wu Jinlu (Sem I & II)

Cynthia He (Sem I)

Liou YC (Sem II)

Department of Biological Sciences

Practical 1 (P-one) Extraction of Genomic DNA from E. coli Extraction of Plasmid DNA

Practical 2 (P-two) Gel Electrophoresis Conjugation Transformation

Practical 3 (P-three) Evaluation of transformation result Evaluation of conjugation result Mutation of yeast (Part A)

Practical 4 (P-four) Evaluation of the results of yeast mutation (Part B)

Dr. Wu JinluDr. He YX/Liou YCLS Lab 1 @ S1A Level 3

Laboratory Sessions

E1 E2

E3

E1

E2

E1

E2

E3

Practical 1 Practical 2 Practical 3

P

-

4

Flowchart of Experimental Objectives

The basic structure of bacterial peptidoglycan and the cell wall structures of Gram-positive and Gram-negative bacteria

Practical 1_@@

http://www.sigmaaldrich.com/technical-documents/articles/biology/glycobiology/peptidoglycans.html

N-acetylglucosamine (NAG) N-acetylmuramic acid (NAM)

Peptidoglycan

Lysozyme

http://bitesizebio.com/2008/02/12/the-basics-how-phenol-extraction-works/

Like dissolves like

immiscible

emulsion of droplets flip inside-out 8-hydroxyquinoline

How phenol works?

Break cell wall with lysozyme

Denature protein and DNA and open the cell wall with SDS (a strong anionic detergent) and NaOH.

Use phenol to extract protein, debris of cell wall and cell membrane, etc

Add sodium acetate to neutralize NaOH

Precipitate DNA from the supernatant with ethanol

Overview on the procedure to extract genomic DNA

Practical 1_@@@

How is plasmid separated from genomic DNA?

Genome extraction: Lysis buffer [2M NaOH and 2% SDS]; mix gently and incubate for 5 min at 37oC.Genomic DNA will be denatured and released into the solution from cell membranes

The critical step: lysing cells with the lysis buffer (SDS and NaOH)

Plasmid extraction : lysis buffer [200 mM NaOH and 1% SDS ] and invert the tube immediately but gently 4-6 times. Vigorous stirring and vortexing must be avoided during lysis.Genomic DNA will be denatured, but will NOT be released into the solution from cell membranes; only plasmid DNA will be released into the solution (supernatant).

In addition:Neutralize (bring down the pH) and adjust the mixture to high-salt binding conditions. The high salt concentration causes denatured proteins, chromosomal DNA, cellular debris, and SDS to precipitate, while the smaller plasmid DNA renatures correctly and stays in solution.

http://www.di.uq.edu.au/sparqmicropipette

Practical 1_@@@@

Using a Micropipette

Grading

Lab Performance and Quizzes 5Gel Electrophoresis (Two-Page Individual report) 4

Experimental Design and Planning for Yeast Mutation (Group work)

2 (bonus allowed)

Describe and Explain the Results of Yeast Mutation (Two-Page Individual report including Part A and B)

4

Laboratory grading (15 points)