Big Bodacious Tips

Big BodaciousTips for faculty and prep staff using the manual

There are lots of tips that instructors will find useful in assisting laboratory preparation and these are detailed in the appendices (especially Appendix XV) and in the Instructors Manual. Here are some particularly useful tips:

1. Obtaining fresh seafood

For those Universities far from the sea, it is important to obtain fresh shrimp for the isolation of bioluminescent bacteria. We routinely order shrimp from the following:

Joe Patti's Seafood
Pensacola, FL
(904) 432-3315

Order shrimp with heads on--they will be freshest and work very well for the isolation. We recommend ordering approximately 25 times more than you need and throwing the extras on the barbie. Here is our recommendation for what to do with the leftovers:

Micro Mike's recipe for spicy grilled shrimp

1/2 lb butter
juice of 2 fresh limes
1-8 dashes of Tabasco
2-4 crushed cloves of garlic
2-4 tsp of cayenne pepper (to taste!)

peel shrimp, place on bamboo skewers, and baste with Mike's special sauce. Grill over very hot coals for 3 minutes per side with additional basting with sauce. Hands used to removing hot media from autoclaves should have no problem turning shrimp.

2. Enzyme recommendations

The key enzyme used in the cloning of the lux operon is Sal I. The quality of this enzyme can vary widely from supplier to supplier and it is important to have good activity, especially in Exercise 8. We have found the Sal I from Gibco-BRL to be of excellent quality and routinely use this supplier.

3. Recipes for Reagent and Media Preparation

The recipies for almost all of the reagents and media required for performing the exercises are located in Appendix XV. The following were omitted or need clarification:

Ethidium bromide-agarose plates (for Exercise 9):

To 240 ml of deionized water add:

2.4 g of LE agarose.
0.6 ml of 1 M MgCl₂

Let the agarose hydrate for 5 to 10 minutes. Heat in microwave until all of the agarose is dissolved. Cool molten agarose to 55C. Add a stir bar and add 12 ul of 10 mg/ml ethidium bromide while mixing. CAUTION: Ethidium bromide is a potent mutagen!!! Mix thoroughly. Pour relatively thin plates (about 15 ml/plate). If the plates are poured too thick, this gives a greater background fluoresence, which will affect the detection of small amounts of DNA. Prepare plates at least one day prior to use. Dry plates at room temperature overnight in a dark place. Wrap in foil and store in the refrigerator.

Please address your questions, comments and suggestions concerning the manual to Mike Winfrey or Marc Rott

Created by Tom Volk

Last Updated: March 22, 1997