Prather:New Student Guide

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Welcome!

Welcome to the Prather Lab! We hope that your stay here will be an enjoyable one. This brief introduction to our lab has been prepared to make your transition into the group a bit easier. It is NOT intended to be an exhaustive working manual, but rather to provide a few bits of information that should help you become familiar with the lab. There is one main rule that you should follow at ALL times in the lab:

DO NOT BE AFRAID TO ASK QUESTIONS!!!!!

The most important aspect of life in the lab is, first, assuring the safety of yourself and your lab mates. The next is respecting the communal nature of the lab. Although there are not very many chemicals in our lab which pose a severe health hazard, there are many pieces of equipment and chemicals which are shared by and necessary to many of us in the lab. Proper use of both will ensure that our lab functions smoothly. If you are unsure how to operate any piece of equipment or you are uncertain about the use or disposal of chemicals, ask your elder lab mates! We would rather spend 50% of our time assisting you in the lab than lose 95% of our time as the result of an unfortunate accident which could have been prevented. (Remember, too, that we all make mistakes, no matter how long we’ve been in the lab. Perfection is neither required nor expected!)

Our lab work includes many different aspects of biochemical engineering, biochemistry, and molecular biology, employing a wide variety of experimental techniques. This guide is meant to highlight only those general issues which will affect the vast majority of students in the lab. Happy reading and once again, welcome to the lab.

Strains and Plasmids

The heart of much of the work in our lab revolves around the use of specific bacterial strains and plasmids. We currently have stocks of over 75 strains. All of the commonly accessible stocks are stored in the -80oC freezer on the top shelf (labeled “Main Storage Box”). Information on each strain is contained in the “Strains Database” file, located on the lab Strainmaster’s computer. The database is searchable by any field within a record, so you can easily search for the location and availability of a strain/plasmid of interest.

In order to ensure that any strains or plasmids that you obtain can continue to be useful to future Prather Lab members, you should be sure to store stocks of them in the freezer and register those stocks in the database.

Making Freezer Stocks

Preparing freezer stocks is simple. Strains should be grown in rich liquid medium to mid-exponential phase. Dilute the cultures 1:1 with sterile, cold 30% glycerol (to ~ 15% glycerol) and transfer 1.8 mL of the mixture to 2 mL cryogenic tubes (so put 900μL cells and 900μL of 30% cold, sterile glycerol in each vial). A total of three tubes should be prepared per strain you wish to store. Each tube should be labeled with the strain’s name, your initials, the date, and tube number (on the cap as well as the tube face). One tube is placed in the “Main Storage Box” on the top shelf of the -80oC freezer while the other two are placed in the “Back-up Storage Box” located on the bottom shelf of -80oC freezer. You must also register each of your frozen strain stocks with the Strainmaster.

These stocks are for long-term storage and should be accessed infrequently. If you will be using a strain repeatedly over several months (or years!), you should prepare additional cryovials to keep in your personal storage box.

Registering Stocks

There is a folder labeled “Completed Frozen Stock Data Sheets,” which is kept on the shelf above the general purpose lab computer. This folder contains information about each strain (box location, strain name, plasmid, characteristics, etc.) in paper form. Similar information is also located in the “Strains Database” group intranet file. There is also a folder in that same location that contains the blank, pre-numbered, data sheets. After strains have been stored properly in the -80oC freezer, a blank form must be filled out and given to the Strainmaster. There are brief instructions on the forms to help you complete them, but be sure to ask for assistance if you are not sure about the proper way to log your strains and plasmids. Plasmids should always be stored within a strain for our stocks. You can also keep plasmid DNA frozen in solution in the stocks, but do not store isolated plasmid DNA in place of transformed bacterial strains.