Molecular Microbiology Laboratory

Instructor:Dr.

Office Hours: by appointment

Teaching Assistants:

Required Texts:

Molecular Microbiology Laboratory by W. Ream, B. Geller, J. Trempy & K. Field,

Academic Press, 2013 (ISBN: 978-0-12-397044-2)

How to Write & Publish a Scientific Paper, 7th Edition by R. A. Day and B. Gastel,

Greenwood Press, 2011 (ISBN: 978-0-39195-8)

Attendance is mandatory.

1 unexcused absence = -5% >2 absences = Incomplete. 15 minutes late = ½ absence

Excused absences considered case-by-case.

You must complete missed assignments.

Please do not attend class if you have a contagious illness.

To request an excused absence, contact me before class.

Final Grade:A = 94-100% of top scoreA- = 90-93%B+ = 87-89%B = 83-86%B- = 80-82%C+ = 76-79%C = 70-75%C- = 65-69%D = 50-65%F = < 50%Lab Reports = 20% each x 4 = 80%Test = 20%

No partial credit for problemsthat involve calculations.

Details are important.

Decorum is important.

End personal conversations when class begins.

Limit conversations to class-related matters.

Attend class on time; stay entire class.

Read assignments & procedures before class.

Bring lab & writing manual to class.

Participate in class writing activities.

Complete assignments on time.

Prepare flow chart before experiment.

You may not begin without a flow chart.

Ask questions if you do not understand.

Sample Flow Chart1 ml O/N cult. strain DH5 in LB

dilute 1 x 10-2

10 ml LB in 125 ml Erlenmeyer

shake 30 C @ 150 rpm

A600 = 0.2

1 ml 9 ml spin 5 min microfuge @ RTstore on ice

dump supernatant pellet

Goals

Teach molecular biology techniques commonly used in life sciences.

Develop scientific writing skills.

Experiment 1:

Plasmid Purification & Restriction Mapping

Experiment 2:

Affinity Purification of Histidine-Tagged Fibronectin-Binding

Protein

Experiment 3:

Polymerase Chain Reaction &

DNA Sequence Analysis of Bacterial Ribosomal RNA Genes

Experiment 4:

Restriction Fragment Length Polymorphism Analysis of

Ribosomal RNA Genes from Uncultured Bacterial

Communities

Wear lab coat and safety glasses.

Do not pipette by mouth.

Do not eat, drink, or chew gum in lab.

Disinfect bench before & after work.

Wash your hands after working.

All bacteria you use may cause disease.

Disinfect contaminated equipment & surfaces.

Place liquid cultures, supernatant solutions & glassware in autoclave containers.

Discard plastic tubes, tips & plates in autoclave bags.

Discard phenol & chloroform in organic waste containers.

Lab coats & safety glasses are REQUIRED when you use phenol.

Do not wear shorts or sandals.

Phenol causes severe chemical burns; wash with water to remove phenol.

Preparing a Lab Report

NameTitle: concise and specific. Date

Purpose: 1-2 sentences. Be specific.

Methods: List procedures used; cite lab manual pages. Note modifications.

Results: Begin with 1-2 sentences outlining experiment.

Describe what you observed.

Do not repeat methods section.

Include graphs, tables, photographs & sequencing electropherograms.

Figures & tables need title, number &legend explaining symbols, etc.

Specify units on axes of graphs.

Label columns and rows in tables.

Number lanes on photographs of gels.

In legend describe contents of lanes.

Place figure ASAP after first cited.

Discussion:Begin with purpose of experiment.

Interpret data in results section.

Discuss meaning of observations.

Mention alternative interpretations; indicate which you think is correct.

Discuss all results, even if unexpected or negative.

If experiment did not work, indicate what went wrong.

Conclusions: Summarize meaning of results; 2-3 sentences.

References: List references used to prepare report.

Questions: Include answers at end of report. Show calculations.

It has been shown that

Helicobacter pylori causes stomach ulcers (1).

It has been shown thatHelicobacter pylori causes stomach ulcers (1).

Helicobacter pylori causes stomach ulcers (1).

Phrases to Avoid Use: instead of:"is" "is known to be""were" "were found to be"

"were observed to be" "were determined to be"

"was" "was identified as being" "it was observed that"

"may be" "is thought to be"

Eliminate: "It was found that" "The results show that" "The purpose of this study was "

An uninformative title:

Terminal Restriction Fragment Length Polymorphism Analysis of 16S Ribosomal RNA Genes from Uncultured Bacterial Communities

An informative title:

Streptococcus, Prevotella, and Veillonella species predominate in the gastric microbiota of patients with inflammatory bowel disease and in control patients

Proposal Abstract

Collect bacteria from environment (p. 51).

You decide what to sample.

Write 1-2 page proposal to justify choice (due class 6).

See lab book for guidelines (xxxiii-xxxvi) & examples (pages 129-133).

Proposals should:

Have a title (see Day, Chapter 7).

Cite peer-reviewed papers.

State question you want to answer.

Indicate why work is important.

Show how work will answer question.

Dr. Ream, I just had to tell you that the textbook (Day & Gastel) we used in 311 came with me to Bermuda, helped me write my CV, my proposal, my progress reports, my proposal to Steve, and my final REU paper and thesis. It is amazingly helpful and I've pretty much read it cover to cover. That doesn't make me a perfect writer! But what a great resource. One of those things Professors always tell you that you don't believe till after the fact. Many thanks, Carmen Denman