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Furman University

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A START-UP MANUAL FOR UNDERGRADUATE RESEARCH STUDENTS IN MICROBIOLOGY
Active Learning from the Very Beginning
Keri Law, Rachel Lamb, and Min-Ken Liao

FROM RESEARCH ARTICLES TO LAB PROTOCOLS

Every experiment must have a protocol which can be obtained from another investigator, a lab manual of protocols, or a "materials and methods" section in published papers. The protocols of the experiments that are routinely performed in this lab can be found in the "Protocol book" on the "catalog" shelf in the lab. Some of the protocols are laminated, which allows you to mark on them. You are encouraged to check each step with a marker to ensure the accuracy in performing each experiment. Erase the marks with 75% ethanol and kimwipes. If you obtain the protocol from a protocol book, make a copy of the procedure and attach it to your labbook. Also, record the name of the book, the authors, and the page number of the protocol as part of the permanent record. What's mentioned above is as straightforward as cooking: Get the recipe from a friend or from a cook book and follow the recipe to prepare your favorite dish. To write up a protocol from a "materials and methods" section in a published paper; however, is more challenging. You have to do some "de-coding". To begin with, you have to bear in mind that people only publish the final concentration of each solution, never the concentration of stock solutions or step-by-step protocol for preparation. Hence, to do an experiment using a published procedure, you have to translate the "method" into a "protocol". Here are two examples for you to practice.

Example 1:

"...Rhodococcus strains were grown at 26ºC in MYP medium containing (gl^-1): glycerol, 10; polypeptone, 5; yeast extract, 3; malt extract, 3; KH2PO4,1.0; K2HPO4 1.0 (pH 7.0)."

What are the ingredients you need to make MYP medium?

The concentration is expressed in "g/1". This means "gram per liter". How many grams of each ingredients will you need to make a liter of MYP? Write a recipe for making MYP medium

How many grams of each ingredients will you need to make 300 ml MYP?

Example 2:

"...Cells were thawed and suspended in 300 ml of buffer A [50 mM sodium phosphate (pH7.0), 5 mM beta-mercaptoethanol]

Let's make buffer A. What are the ingredients you will need?

What are the final concentrations of each?

How to make a 100 ml 0.5 M sodium phosphate monobasic (pH 7.0) stock solution? Show your calculation.

To make 300 ml of buffer A, how many milliliters of 0.5 M sodium phosphate monobasic will you need? Show you calculation.

Beta-mercaptoethanol comes in liquid form and you need to know the density to figure out the molarity. On the bottle you can find information on molecular weight (FW) and purity, but not the density. However, you can find it in the catalog of the supplier. We ordered our chemicals from Sigma. The easiest way to find beta-mercaptoethanol in the catalog is by using the "product number", which can be found in the upper left corner of the label. In this case, the product number for beta-mercaptoethanol is __________. At the end of the Sigma catalog, there is a section called "product index." Find (product number of beta-mercaptoethanol) and it will tell you in what page you can find the product. In our case, beta-mercaptoethanol can be found in page _________. And right there it tells you that the density of beta-mercaptoethanol is __________ g/ml. Our bottle has 100 ml in it. How many grams of beta-mercaptoethanol are there in one bottle? Show your calculation.

You know the molecular weight of beta-mercaptoethanol. Determine how many moles of beta-mercaptoethanol in one bottle. Show your calculation.

You know that there is 100 ml in the bottle. What is the molarity? Show your calculation.

Now you know the stock concentration. Let's go back the our "methods" section. The recipe calls for 50 mM. If you want to make 300 ml of buffer A, how many milliliters of beta-mercaptoethanol will you need? Show your calculation.

For 300 ml of buffer A we need _____________ ml of 0.5 M of phosphate buffer and _________ml of beta-mercaptoethanol. How many milliliters of dH2O will you need to make up to 300 ml of buffer A?