Alta.biology.utah.edu

A. LB Plates 500 mL (20 - 25 plates)
1.
Combine 2 large capsules of LB media, 7.5 g agar, a large magnetic stir bar and 500 mL water ina 1 L Erlenmyer flask without baffles. (Alternatively, substitute ready made LB+Agar capsules -recipe with number of capsules to use is on the container).
Use a small piece of heat-indicator tape to secure a piece of foil over the mouth of the flask.
Autoclave 121 °C for 40 minutes in a pan of shallow water.
Mix by stirring on a magnetic stir plate and allow to cool to ~50-60 °C.
Clean off a generous bench area and sterilize with ethanol.
When the temperature has reached ~55 °C (can handle flask without gloves), add antibiotics or
other heat sensitive additives and continue stirring until these are dissolved. Note: DO NOT stir
too vigorously since bubbles will cause problems later.
Remove sterile plates from their sleeve and arrange in stacks of 5 plates per stack. Save thesleeve to store the plates once they have cured. Label with color-coded stripes (Table 1.1), Remove the foil cover from the media and flame the mouth of the flask. Beginning with the bottomplate, lift off the lid and the empty plates above, pour ~22 mL media into the plate, swirl gently tocover the plate evenly, and replace the lid. Repeat for the next plate and so on until all plates inthe stack have been poured. Flame the mouth of the flask before pouring the next stack of plates.
Bubbles can be removed by flaming the surface of the plate.
Rinse the flask immediately after pouring the last plate and before the residual agar sets.
Group the plates close together and cover loosely with paper.
Allow plates to cure for 2-3 days and then stored at +4 °C, with label tape holding the bag closed.
Table 1.1 Color code for antibiotics in LB plates B. SOB Media
12.
Combine tryptone, yeast extract, 5 M sodium chloride as indicated in Table 1.2.
Use a small piece of heat-indicator tape to secure a piece of foil over the mouth of the flask.
Autoclave 121 °C for 40 minutes in a pan of shallow water.
Prior to use, add sterile filtered 1 M magnesium sulfate to a final concentration of 0.02 M Table 1.2 SOB media for 50 mL and 200 mL cultures Autoclave and cool to room temperature, then add heat sensitive ingredients1 M MgSO4 Antibiotic Additives (if any)25 mg/mL Amp *Prepare 50 mL in a 250 mL baffle flask.
‡Prepare 200 mL in a 500 mL baffle flask.
C. LB & 2xYT*
18.
Combine 2xYT capsules and water as indicated in Table 1.3. For LB media use 1/2 as manycapsules Use a small piece of heat-indicator tape to secure a piece of foil over the mouth of the flask.
Autoclave 121 °C for 40 minutes in a pan of shallow water.
Prior to use, add sterile filtered 1 M potassium phosphate pH 7.8 to a final concentration of 30 mMand sterile filtered 1 M Glucose (Dextrose) to a final concentration of 5 mM.
Table 1.3 2xYT* media for 60 mL, 500 mL, and 750 mL cultures Autoclave and cool to room temperature, then add heat sensitive ingredients1 M potassium phosphate pH Antibiotic Additives (if any)25 mg/mL Amp 1 Prepare 60 mL in a 250 mL baffle flask.
2 Prepare 500 mL or 750 mL in a 2 L baffle flask.
D. Table 1.4 Antibiotic stock and working solutions
Ampicillin is prepared from the free acid or sodium salt dissolved in water. To make 40 mL of a 25
mg/mL solution, dissolve 1 g ampicillin in a final volume of 40 mL. If using the free acid, add 50 µL
aliquots of 5 N sodium hydroxide until the suspension clears. Dispense as 1 mL aliquots and store -20
°C. Working concentration is 125 µg/mL in plates and starter cultures and 500 µg/mL in large volume
cultures. Take care to not allow cultures to overgrow since the enzyme that confers resistance to
ampicillin is exported to the periplasm and leaks into the media.
Kanamycin is dissolved directly in water. To make 40 mL of a 10 mg/mL solution, dissolve 0.4 g
kanamycin sulfate in a final volume of 40 mL. Dispense as 1 mL aliquots and store -20 °C. Working
concentration is 40 µg/mL.
Chloramphenicol is dissolved in ethanol. To make 40 mL of a 34 mg/mL solution, dissolve 1.36 g of
chloramphenicol in 40 mL of ethanol. Store -20 °C. Working concentration is 17 µg/mL.
Tetracycline is dissolved in ethanol. To make 40 mL of a 5 mg/mL solution, dissolve 0.2 g of tetracycline
in 40 mL of ethanol. Store -20 °C. Tetracycline is light sensitive. Working concentration is 12.5 µg/mL.

Source: http://alta.biology.utah.edu/protocols/001_media.pdf