BIO 143 Week 5 Part 1 - Fall 2018 PDF

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Week 5 Part 1 Lecture notes...

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BIO 143 - SEA PHAGES Laboratory II Week 5 Part 1 Toolbox of Miscellaneous Methods Making Top Agar (1X Middlebrook Top Agar for M. smegmatis) Objective ● To make molten agar/media mixture for growing bacterial lawns on agar plates

Rationale ● Mixing a small amount of bacteria with molten top agar and spreading it over the surface of an agar plate results in a smooth lawn perfect for viewing bacteriophage plaques. ○ When working with M. smegmatis ■

top agar is made by mixing 2X Middlebrook Top Agar with an equal volume of 7H9 liquid media.

Supplies ● 100mM CaCl2 (sterile) stock (1 ml) ● 7H9 liquid media neat (50 ml aliquot) ● 2X top agar (50 ml aliquot)

Procedure ● A. Prepare your bench for aseptic work and assemble your supplies. ● B. Prepare 7H9 media. ○ 1. Using aseptic technique add 1 ml of CaCl2 1. to a 50 ml aliquot of 7H9 neat media. ○ 2. Place in 55 °C bath.

● C. Melt the 2X top agar in a microwave. ○ 1. Make sure the cap is loose! ○ 2. The 2X top agar should come to a boil but not boil over. ○ 3. The agar must be completely melted, so carefully swirl the bottle and check to make sure there are no clumps. ○ 4. Once the agar is completely melted, place the 2X top agar in a 55 °C bath. ● D. Aseptically combine the hot 2X top agar and 7H9/CaCl2 D. to make 1X top agar. ○ 1. Keep at 55 °C until use.The CaCl2 ○

2. will precipitate out of solution and form a hazy layer on the bottom of the bottle. ■ It will go back into solution when swirled....