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-	-Make plates with a thick layer of medium, 100 ml medium per big Petri dish.Medium of Streptomyces is mannitol soya flour medium (MS or SFM) 300 ml per 1L bottle to avoid boiling over in autoclave
-	*Agar                          6 g	+	'''Concentrations for antibiotics '''
-	*D-mannitol                    6 g	+
-	*Soya flour                    6 g	+
-	*Demiwater                      300ml	+
-	*Plating ΔrdeAB spores          10^5 /plate	+
-	-Incubate for 7 days at 30 °C	+	Antibiotic                 Stock                         E.coli                         B.subtilis
-		+	*Ampicillin (Amp) (in H2O)   50 mg/ml (4 °C) 100(μg/ml) (:500)
-	-Harvest mycelium with razorblade	+	*Kanamycin (Km)           10 mg/ml (4 °C) 20 (μg/ml) (:500)                        10 (μg/ml) (:100)
-		+	*Spectinomycin (Spec) (in H2O)   10 mg/ml (4 °C) 100(μg/ml) (:100)                 100 (μg/ml) (:100)
-		+	*Erythromycin (Ery) E.coli stock  10 mg/ml (-18 °C) 150 (μg/ml) (:66.6)
-	'''Expression Chaplins in NZ8900'''	+	*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C)                                 2-5 (μg/ml)
-		+	*Tetracyclin (Tet) (50% EtOH)     3 mg/ml (-18 °C) 12 (μg/ml) (:250)                 6(μg/ml) (:500)
-	-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)	+	*Chloramphenicol (Cm) (50% EtOH)    5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333)         5(μg/ml) (:1000)
-		+	*Lyncomycin                   12.5 mg/ml (4 °C)                                 12.5 (μg/ml) (1:1000)*
-	-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)	+	*Hygromycin                   50 mg/ml (4 °C)                                 125 (μg/ml) (:400)
-		+
-	-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1	+
-		+
-	-Grow till OD600 is approx. 0.5	+
-		+
-	-Take 2 ml t=0 sample	+
-		+
-	-Induce with 1% subtiline	+
-		+
-	-Continue growing	+
-		+
-	-Take 2 ml samples every hour	+
-		+
-	'''Sample treatment'''	+
-		+
-	-Take 2ml sample and spin down	+
-	'''Supernatant'''	+
-	-1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)	+
-		+
-	-Do TCA precipitation until drying pellet after washing with aceton	+
-		+
-	-Continue with TFA treatment	+
-		+
-	'''Cell pellet'''	+
-		+
-	-TFA treatment	+
-		+
-	-Pellet of TFA treatment with cells should be airdried	+
-		+
-	-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)	+
-		+
-	-Speedvacum	+
-		+
-	-TFA treatment	+

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Latest revision as of 17:19, 9 September 2010

Concentrations for antibiotics

Antibiotic Stock E.coli B.subtilis

• Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500)
• Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)
• Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)
• Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6)
• Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)
• Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)
• Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)
• Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*
• Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)