• Agarose Gel Electrophoresis
• Bacterial Streak Plate
• Bacterial Transformation
• DNA Ligation
• PCR
• Pipettors
• Plasmid DNA Isolation and Restriction Enzyme Digests
• Preparation of Agar Plates

• Laboratory Citizenship & Performance
• BioSciences Lab Program Honor Code
• Molecular Biology Tips
• Writing Up Methodology

• *LAB SAFETY*
• Laboratory notebook

• Biuret Protein Assay
• Experimental Biosciences

Preparation of Agar Plates

Growth and Check of Bacterial Strains

Bacteria can be propagated on liquid or solid media. The use of liquid allows large quantities of bacteria to be harvested but does not permit easy selection or determination of phenotype of single cells. The technique of "streaking" cells onto a solid media provides simple isolation of colonies arising from single cells. Colonies selected for the desired phenotype are then used to inoculate liquid broth. A single colony inoculum is preferred because bacteria can undergo many types of mutations naturally. The instability of some of the mutations, especially transposons and phages, can allow some cells to lose characteristics important to the selection scheme and may complicate the analysis. It is always wise to check the parent strain for proper phenotype that reflects the genotype and then use "picks" from single colonies to start liquid cultures.

Luria-Bertani (LB) plates with the appropriate antibiotic for selection will be prepared for plating transformations.

NOTE: LB agar has been prepared for you; the information about the media is included here so you will know the contents.
Recipe for Luria-Bertani (LB) Agar (per liter):
1% Bacto-Tryptone = 10 g
0.5% Yeast extract = 5 g
85 mM NaCl = 5 g
1.5% Bacto Agar = 15 g

*adjust pH to 7.0 with NaOH and bring to 1 L; sterilize by autoclaving for 20 minutes at 121°C at 15 psi
**autoclaved media is always cooled to 50-60°C prior to the addition of antibiotics and some salts that are inactivated or precipitated by autoclaving; holding the agar at this temperature prevents congealing

Sterile Technique

Media can be contaminated by contact with non-sterile surfaces or by air borne organisms. Remove lids and coverings carefully avoiding contact with any part of the cover that may contact the media. Lids and coverings should be held with media side down at all times. Air borne contaminants are usually falling downward. Replace the coverings carefully so that the rim of the container makes contact only with sterile surface of the inside of the cap. The use of a flame helps maintain aseptic materials. Working near a flame can decrease air borne contamination. The flame is also used to singe surfaces to maintain sterility. The mouth of the tube or flask is passed through the flame before and afer pouring. The cap or cover is also passed through the flame prior to replacing on the container.
Caution: The flame is used to singe the surfaces only. Do not hold the items in the flame to make them hot. Glass flasks, even Pyrex, can break from the hear or when the cooler medium hits the hot surface.

PROTOCOL (work as a team)

1. Stack 4 Petri dishes (right-side up)
2. Turn on gas halfway and light Bunsen burner with striker
3. Swirl large flask of LB-agar held at 50-60°C to mix contents well
4. Transfer ~100 ml of LB-agar to a sterile flask and add the appropriate antibiotic stock in a 1:1000 ratio (1 µl of antibiotic for every ml of media); mix by gently swirling but avoid creating bubbles and keep the media sterile
   ***Use sterile technique - don't contaminate the media***
5. Using sterile technique, pour 20-25 ml of LB-agar into a Petri dish (enough to cover the bottom of the dish)
   • Work rapidly to pour the plates; the agar will begin to congeal in 5 minutes with the flask at room temperature
   • Stacking the plates while pouring saves bench space and is accomplished by lifting the stack of 3 empty plates by the lid of the bottom most plate. Agar is poured into the Petri dish and the stack is replaced; move your hand to the second lid and lifte the stack again to pour the second dish of agar; repeat until all the plates in the stack are poured
   • Bubbles on the surface of the media can be eliminated by passing the flame quickly over the bubbles
6. Turn off the gas!
7. Pour any remaining media in the trash can and rinse out flask with RO water
8. Let the plates sit at room temperature until they are congealed (30-45 minutes) and store in plastic and inverted at 4°C

We would like to thank New England Biolabs for their generous support of our laboratory program