BISC209/S11: Assignment 209 Lab5

Materials and Methods

Due at the beginning of Lab 6.
How to Write a Materials and Methods Section
It can be a daunting task to write a single paper on several months of experiments, particularly when those experiments result in a huge amount of disparate data. Turning an entire semester of lab work into a Materials and Methods section for your final paper can be particularly overwhelming, but it need not be. It is less so if you have kept a good lab notebook that contains all the information necessary to explain how to make or purchase reagents and clearly explains how you used these materials to accomplish your small and large goals.

It is also much easier if you think of your semester's work as a whole comprising many smaller, independent parts. Organization of your M&M section by goal and by independent tools is MUCH better than trying to write a chronological description of everything you did each day. A time-course description of the semester is NOT the way to organize and report the materials and methods applied to this project. The focus in such a description is on you and what YOU did. The focus of a good M&M section is on how each of the stated goals was accomplished. The wiki protocols tell you exactly what to do in what order, recognizing that you are new to most of these tools and materials. The detail in these protocols is far beyond anything needed in M&M and the focus is wrong. It would make your M&M section unreadable and unintelligible if you were to include all the wiki's trivial directives about what to do to tubes and plates at each step of every protocol. The wiki also lacks, in these detailed chronological lab day descriptions, lots of crucial information that you need in M&M. The wiki, largely, ignores all the behind the scenes work required to make or acquire the materials necessary to accomplish the work described. The ingredients and concentration information needed to make media,etc. or the information required to order reagents and use them properly must be included in a M&M section. Fortunately, if all that information has been previously published or can be found on a kit or reagent suppliers web-site, you need not repeat it-- as long as you include sufficient information in your M&M for a reader to find the supplier or manufacture's product information or you have the full citation to the paper or methods manual that describes how to make all the materials and to perform the testing. You MAY NOT cite the wiki for any information.

To help you with this assignment we have provided an organization scheme for your M&M section and have written a few sample sections for you. The best way to learn how to write M&M properly is to look at published papers that use similar tools and see how they described the use of those tools. If you find a reference to a publication that may describe a protocol you performed, you must look up that reference and be sure they did the procedure the way you did and, if there are slight modifications, describe them.

How to Hierachially Organize the M&M section of this project?
First describe any procedures common to the three basis divisions: Each should be separately titled. If you did them more than once, there is no need to mention that. Separately title each protocol, stressing the goal rather than the tool in the title. Order them by deciding which goal must be accomplished first. (Note that you do NOT have to write up protocols that you have not performed yet. This list is to help you know what to include in your final M&M draft.) Acquiring a Soil Sample for Bacterial Community Identification and Assessment Making a Soil Extract

After describing general protocols common to the three lines of our investigation, organize by those 3 lines and then organize specific protocols under each of these sub-headings:

1. Culture-Independent Identification of Bacteria in a Soil Community by 16s rRNA Gene Sequencing of whole soil genomic DNA(notice goal is before tool)
   1. Isolation of genomic DNA from soil
   2. Amplification of 16s rDNA by polymerase chain reaction. This protocol should include the adjustment of DNA concentration, the pcr product "cleanup" and the assessment of success by agarose gel electrophoresis). The final goal of this protocol is to end up with lots of copies of double-stranded DNA amplified from the 16s rRNA gene template found in as many as possible of the bacteria in your soil community.
   3. Separating the 16s rRNA genes of bacteria in a soil community. This protocol should in include cloning the 16s rDNA into a plasmid vector; transforming chemically competenet E. coli with the clones; selecting for transformants with a plasmid containing a soil community bacteria's 16s rRNA gene; preparing glycerol stocks of successful, cultured transformants; DNA sequencing of 16s rRNA genes from clones (name the sequencing method, facility and location we will use)
2. Culture-Dependent Soil Microbial Community Community Physiological Profiling
   1. Carbon source utilization: average metabolic response (AMR) and community metabolic diversity (CMD)
   2. Nitrogen cycling: Most Probable Number (MPN) of Ammonia, Nitrate, and Nitrite producers
   3. Exoenzymes: Cellulase, Amylase, Phosphate Solubilization
3. Culture-Dependent Assessment of Functional Roles and Relationships in Selected and Isolated Bacteria from a Soil Community
   1. Isolation of Methylotropic Denitifiying Bacteria to Pure Culture
   2. Isolation of Azotobacter Bacteria to Pure Culture
   3. Antibiotic Production Testing
   4. Quorum Sensing Assessment
   5. Mutualistic and Antagonistic Interaction Assessement

General tips:
Please look at the Methods sections of published scientific research papers (there are many in the References tool in Sakai) to see how the protocols in this wiki differ from Materials and Methods descriptions in a scientific paper. Notice that M&M can be very short, particularly if you have used a kit or a previously published protocol for a part or all of the experiment. If some part of your work is published elsewhere, such as in a journal article's methods section or in a kit manufacturer's website, you may reference the kit manufacturer or use a journal article citation to the previously published directions.

Remember that all separately sectioned protocols are independent; therefore, you can not assume your reader knows what soil sample or extract you mean unless you say, "soil sample obtained as described in Materials and Methods".

NEVER, EVER use the word "tube" in Materials in Methods. We often tell you, in a protocol, to do ____ to the "tubes". In the context of a lab manual procedure description, that's fine. For Materials & Methods, you must be more specific. It's what's happening to the materials in the tubes that is important. Tubes and plates are just pieces of plastic or glass. Your methods section should not read like a series of "did this, then did that" descriptions, but should, instead, describe how you acquired and processed the starting material to end up with the ending material.

In order to write about how you accomplished your experimental goals succinctly and clearly, you must thoroughly understand what part of your goal is accomplished in each step. It does not require thorough understanding to follow the directions in the wiki and end up with a successful experiment; however, to write about your experiments in such a way that someone who is not part of this course can understand what you did and how it contributed to your overall goal... that's is not so easy. If you make sure that everytime you leave lab you understand how each part of each day's work fits into the project's goals, writing M&M, as well as the other parts of this paper, will be manageable.

Keep in mind that when you reference a reagent manufacturer's web site (such as the New England BIolab's website directions for using Phusion® High-Fidelity PCR Master Mix with HF Buffer-- the reagent we used to amplify our target DNA by pcr (found at: | http://www.neb.com/nebecomm/ManualFiles/manualF-531.pdf), the manufacturer does not include the sequences of the primers we used (since those are unique to your target DNA) nor the exact thermal cycler program (since that is tweaked for the length of the DNA section you want to copy and for the relative CG content of the template DNA). Therefore, you must give more than a brief citation and web address. In the case of the pcr amplification using a commercial master mix, you must be specific about the template DNA, the primer information for amplifying 16s rDNA, and give the exact thermal cycler program you used.

There is an extensive handout on writing a Methods section in the Resources section of this wiki.