Media

LB liquid medium for E. coli.

In large beaker, combine:

10 g tryptone

5 g yeast extract

10 g NaCl

(alternatively, use 25 g of premixed LB powder instead of tryptone, yeast extract and NaCl)

1 L deionized water

use stir bar to mix completely, pour into glass bottles and autoclave 15’ to sterilize. Make sure lids are on loosely when autoclaving and tighten after media has cooled.

LB plates:

In 2 liter flask combine:

10 g tryptone

5 g yeast extract

10 g NaCl

15 g agar

1 L deionized water

stir bar

autoclave to sterilize, stir for 30’-60’ to cool or put flask into 50 C water bath for 30’ or more to cool (agar won’t solidify at 50 C so you can leave it in water bath for longer periods), add antibiotics if needed (see below), and pour into sterile petri dishes (approx. 20 ml/plate).

Synthetic Complete Dextrose (SCD) plates (amino acid drop-out media for yeast)

    In 2 liter flask, combine:

• 1.7 g Yeast Nitrogen base
• 5 g Ammonium sulfate
• 0.6-1.6 g amino acid mix (type and amount depends on which amino acid is to be left out)
• 20 g dextrose
• for making plates, add 20 g agar
• 1 liter deionized water
• a stir bar

Mix the ingredients and then autoclave 20 minutes on liquid cycle. For liquid media, autoclave in bottles with loose tops.

YePD for yeast (aka, YPAD or YPDA)

In 2 liter flask combine:

· 10g yeast extract

· 20g peptone

· 20g dextrose (glucose)

· 20g agar (for plates

· optional: add 100 mg adenine – required for adenine mutants.

· add 1 liter deionized H₂O, stir to mix

autoclave for 20 min on liquid cycle

for liquid media, autoclave in bottles with loose tops.

1/2X MS media for Arabidopsis

• 2.2 gm MS powder
• 10 g sucrose (1% final)
• water to 1 liter
• mix with stir bar
• pH to ~5.7 with 1 M KOH (usually not required, but check)
• add 7 gm phytoagar (not regular agar)

• Autoclave 15-20’; stir 30-60 minutes to cool (can place flask in 50 C water bath to cool- agar won’t solidify).
• Add antibiotics/herbicides at this point, if required.
• optional: add vitamin mix to 1X from 1000X stock

• pour plates (make them thicker than usual so roots can grow better)

Antibiotics

	Working Concentrations (ug/ml)			Stock	Solvent
	E.Coli	Agro.	Plants	mg/ml
Ampicillin	50-100	50-100	-	50	H2O
Kanamycin	50	20-100	75-2000	50	H2O
Tet	50	3-20	-	10	EtOH
Hyg	10-50	10-50	10-400	10	H2O
Rif	-	10-150	-	20-50	MeOH
Sulfa*			7.5	7.5	H2O
BASTA			10 (agar), 120 (spray)		H2O
Chloramphenicol	170			34	ETOH
streptomycin	50			10	H2O

*Na-Sulfadiazine (mw = 272) Sigma S-6387; Fisher does not carry this – I bought salt-free version from fisher; salt-free sulfadiazine is mostly insoluble so I add the powder to 1 ml ethanol and then add directly to the autoclaved media solution while stirring just prior to pouring plates.