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Team:Groningen/Growing Streptomyces
From 2010.igem.org
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-TFA treatment | -TFA treatment | ||
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| + | '''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) | ||
Revision as of 23:48, 24 August 2010
-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
- D-mannitol 6 g
- Soya flour 6 g
- Demiwater 300ml
- Plating ΔrdeAB spores 10^5 /plate
-Incubate for 7 days at 30 °C
-Harvest mycelium with razorblade
Expression Chaplins in NZ8900
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)
-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
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)
