http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Neima2010.igem.org - User contributions [en]2024-03-29T13:20:32ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/SMG17MCSMG17MC2010-10-27T20:59:04Z<p>Neima: </p>
<hr />
<div>'''SMG17MC'''<br />
<br />
-M17<br />
<br />
-0.5 M Sucrose <br />
<br />
-0.5 % Glucose<br />
<br />
-20 mM MgCl2 <br />
<br />
-2 mM CaCl2</div>Neimahttp://2010.igem.org/GSM17-_AgarGSM17- Agar2010-10-27T20:58:20Z<p>Neima: New page: '''GSM17- Agar''' - M17-Agar - 0.5 M Sucrose - 0.5% Glucose</p>
<hr />
<div>'''GSM17- Agar'''<br />
<br />
- M17-Agar<br />
<br />
- 0.5 M Sucrose<br />
<br />
- 0.5% Glucose</div>Neimahttp://2010.igem.org/SMG17MCSMG17MC2010-10-27T20:56:27Z<p>Neima: New page: '''SMG17MC''' -M17 -o.5 M Sucrose -0.5 % Glucose -20 mM MgCl2 -2 mM CaCl2</p>
<hr />
<div>'''SMG17MC'''<br />
<br />
-M17<br />
<br />
-o.5 M Sucrose <br />
<br />
-0.5 % Glucose<br />
<br />
-20 mM MgCl2 <br />
<br />
-2 mM CaCl2</div>Neimahttp://2010.igem.org/Team:Groningen/Protocols_for_LactococcusTeam:Groningen/Protocols for Lactococcus2010-10-27T20:54:32Z<p>Neima: </p>
<hr />
<div>'''Preparation of cells'''<br />
<br />
*10 ml ON culture ([[SMGG]]) in 100 ml SMGG<br />
*Grow to OD600=0.2-0.7<br />
*Wash three times with 50 ml icecold [[wash buffer]]<br />
*Resuspend in 1 ml wash buffer<br />
<br />
'''Electroporation'''<br />
<br />
*1 μl DNA in 40 μl cell-suspension in ice cold cuvette<br />
*Electroporate at 2.5 kV, 25 μF, 200 Ohm.<br />
*Add 4 ml [[SMG17MC]]<br />
*Incubate 2 hours (Ery induction=50 ng/ml)<br />
*Concentrate cells to 0.5 ml.<br />
*Plate on [[GSM17- Agar]].</div>Neimahttp://2010.igem.org/Wash_bufferWash buffer2010-10-27T20:53:35Z<p>Neima: New page: '''Wash buffer''' -0.5 M Sucrose (17g/100ml) -10% gylcerol( 11.5ml/100 ml)</p>
<hr />
<div>'''Wash buffer'''<br />
<br />
-0.5 M Sucrose (17g/100ml) <br />
<br />
-10% gylcerol( 11.5ml/100 ml)</div>Neimahttp://2010.igem.org/SMGGSMGG2010-10-27T20:51:23Z<p>Neima: New page: '''SMGG''' -M17 -0.5 M Sucrose -0.5% Glucose -1% Glycine</p>
<hr />
<div>'''SMGG'''<br />
<br />
-M17<br />
<br />
-0.5 M Sucrose<br />
<br />
-0.5% Glucose<br />
<br />
-1% Glycine</div>Neimahttp://2010.igem.org/Team:Groningen/Protocols_for_LactococcusTeam:Groningen/Protocols for Lactococcus2010-10-27T20:49:41Z<p>Neima: New page: '''Preparation of cells''' *10 ml ON culture (SMGG) in 100 ml SMGG *Grow to OD600=0.2-0.7 *Wash three times with 50 ml icecold wash buffer*Resuspend in 1 ml wash buffer '''Electr...</p>
<hr />
<div>'''Preparation of cells'''<br />
<br />
*10 ml ON culture ([[SMGG]]) in 100 ml SMGG<br />
*Grow to OD600=0.2-0.7<br />
*Wash three times with 50 ml icecold [[wash buffer]]*Resuspend in 1 ml wash buffer<br />
<br />
'''Electroporation'''<br />
<br />
*1 μl DNA in 40 μl cell-suspension in ice cold cuvette<br />
*Electroporate at 2.5 kV, 25 μF, 200 Ohm.<br />
*Add 4 ml [[SMG17MC]]*Incubate 2 hours (Ery induction=50 ng/ml)<br />
*Concentrate cells to 0.5 ml.<br />
*Plate on [[GSM17- Agar]].</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-10-27T19:56:52Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Transformation to E. coli | Transformation to ''E. coli'']]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]<br />
<br />
[[Team:Groningen/Chaplins | Chaplins]]<br />
<br />
[[Team:Groningen/Protocols for Streptomyces | Protocols for Streptomyces]]<br />
<br />
[[Team:Groningen/Protocols for Lactococcus | Protocols for Lactococcus]]<br />
<br />
[[Team:Groningen/Antibiotics and Concentrationas | Antibiotics and Concentrations ]]</div>Neimahttp://2010.igem.org/Team:Groningen/Protocols_for_StreptomycesTeam:Groningen/Protocols for Streptomyces2010-10-25T16:19:16Z<p>Neima: </p>
<hr />
<div>'''Growth condition'''<br />
<br />
-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
----<br />
<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
<br />
*Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
<br />
*Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
<br />
*Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
<br />
*Press the loading cilinder on the tower tightl (don't be to careful)<br />
<br />
*Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
<br />
*Put on the nozzle and the lit of the nozzle, tightly<br />
<br />
*The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
<br />
*Adjust your pressure to 13 kpsi<br />
<br />
*Use the pin to start the machine, after two loud klblam!'s remove it<br />
<br />
*Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
<br />
*Run it through another 5X <br />
<br />
*Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
<br />
*Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
-Spin off, 4000 rpm, 5 minutes: Use pellet<br />
<br />
-Cook in 2% SDS<br />
<br />
-Spin off,4000 rpm, 5 min: Use pellet<br />
-Wash 10X with demi-water:<br />
<br />
*Resuspend <br />
*Spin off: Use pellet<br />
*Etc.<br />
<br />
-Freeze pellet (For freeze drying: free it well)<br />
<br />
-Freeze dry your pellet<br />
*Freeze the jar in which you will freeze dry your sample as well<br />
*Turn on the machine, it needs to get on temperature and suck vacuum<br />
<br />
Make sure all the valves are closed<br />
The big red valve needs to be open<br />
The machine is ready when temperature reaches -80 and stays stable<br />
<br />
*Your greiner tube will be put in the jar with either a loose tube hood or parafilm to seal it off (prick 4 holes), seal the jar of with the rubber top<br />
<br />
*Place the jar on one of the valves and open it up slowly<br />
<br />
*When the sample is dry-freezed<br />
<br />
Close the valve and remove the jar<br />
Turn off the machine<br />
Close the red valve</div>Neimahttp://2010.igem.org/Team:Groningen/Antibiotics_and_ConcentrationasTeam:Groningen/Antibiotics and Concentrationas2010-10-25T15:44:21Z<p>Neima: New page: '''Antibiotics''' Ampicillin 100 mg/ml Ampicillin (1000x) Stock *1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH) *Add NaOH or KOH to allow the Ampicillin to dissol...</p>
<hr />
<div>'''Antibiotics'''<br />
<br />
[[Ampicillin]]<br />
<br />
100 mg/ml Ampicillin (1000x) Stock <br />
*1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)<br />
*Add NaOH or KOH to allow the Ampicillin to dissolve <br />
*Filter sterilize 0.2 μm filter and aliquot <br />
*Store -20 °C<br />
<br />
[[Chloramphenicol]]<br />
<br />
35 mg/ml Chloramphenicol (1000x) Stock <br />
*0.35 g in 10 mL 100% EtOH <br />
*Filter sterilize 0.2 μm filter and aliquot <br />
*Store -20 °C<br />
<br />
[[Kanamycin]]<br />
<br />
50 mg/ml Kanamycin (1000x) Stock<br />
*500 mg in 10 mL demi water <br />
*Filter sterilize 0.2 μm filter and aliquot <br />
*Store -20 °C</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-10-25T15:44:01Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Transformation to E. coli | Transformation to ''E. coli'']]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]<br />
<br />
[[Team:Groningen/Chaplins | Chaplins]]<br />
<br />
[[Team:Groningen/Protocols for Streptomyces | Protocols for Streptomyces]]<br />
<br />
[[Team:Groningen/Antibiotics and Concentrationas | Antibiotics and Concentrations ]]</div>Neimahttp://2010.igem.org/Team:Groningen/Antibiotics_Concentrationas_and_OrganismsTeam:Groningen/Antibiotics Concentrationas and Organisms2010-10-25T15:42:46Z<p>Neima: </p>
<hr />
<div>'''Antibiotics'''<br />
<br />
[[Ampicillin]]<br />
<br />
100 mg/ml Ampicillin (1000x) Stock <br />
*1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)<br />
*Add NaOH or KOH to allow the Ampicillin to dissolve <br />
*Filter sterilize 0.2 μm filter and aliquot <br />
*Store -20 °C<br />
<br />
[[Chloramphenicol]]<br />
<br />
35 mg/ml Chloramphenicol (1000x) Stock <br />
*0.35 g in 10 mL 100% EtOH <br />
*Filter sterilize 0.2 μm filter and aliquot <br />
*Store -20 °C<br />
<br />
[[Kanamycin]]<br />
<br />
50 mg/ml Kanamycin (1000x) Stock<br />
*500 mg in 10 mL demi water <br />
*Filter sterilize 0.2 μm filter and aliquot <br />
*Store -20 °C</div>Neimahttp://2010.igem.org/Team:Groningen/Antibiotics_Concentrationas_and_OrganismsTeam:Groningen/Antibiotics Concentrationas and Organisms2010-10-25T15:23:03Z<p>Neima: </p>
<hr />
<div>'''Concentrations for antibiotics ''' <br />
<br />
'''Antibiotic'''''' Stock''' ''' E.coli ''' ''' B.subtilis'''<br />
<br />
*[[Ampicillin]] (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
<br />
*[[Kanamycin]] (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
<br />
*[[Chloramphenicol]](Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)</div>Neimahttp://2010.igem.org/Team:Groningen/Normal_SDS-PAGE_using_Tris-Glycine_Gels_and_Electrophoresis_BufferTeam:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer2010-09-09T17:22:47Z<p>Neima: </p>
<hr />
<div>'''Wear gloves at all times!''' <br />
<br />
Requirements:<br />
*30% acrylamide(Biorad) '''NEUROTOXIN!'''<br />
*4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8<br />
*4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol<br />
*TEMED( in yellow cabinet )<br />
*10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge<br />
<br />
'''Hoeffer Mighty gel system'''<br />
Casting of the gel:<br />
*Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)<br />
*Clean the plates etc with soap, rinse with demiwater and ethanol, and dry<br />
*Assemble the system in the gel casting holder. Mark the line of separation/stacking<br />
*Mix the separation gel in 10 ml plastic tube:<br />
'''for 2 gels'''<br />
'''Seperation gel (0.75 mm)''' ''' 12.5%''' '''16%''' <br />
30% acrylamide 4 ml 5.1 ml<br />
4X separation buffer 2.4 ml 2.4 ml<br />
MQ 3.2 ml 2.1 ml<br />
10% APS 28 μl 28 μl<br />
TEMED 28 μl 28 μl<br />
*Pipet the separation gel mix immediately in between the glass plates until the marked line is reached<br />
*Pipet water-saturated isobutanol on top of the polymerizing separation gel<br />
*Let the separation gel polymerize completely before preparing the stacking gel<br />
*When the gel is polymerized, discard the isobutanol and wash the gel with water<br />
*Mix the stacking gel in a 10 ml tube:<br />
'''for 2 gels'''<br />
mQ 1.92 ml<br />
4X stacking buffer 0.83 ml<br />
30% acrylamide 560 μl<br />
10% APS 14 μl<br />
TEMED 7 μl<br />
*Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker<br />
*Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube<br />
*Disassemble the gel casting holder and take out the gel/plates<br />
*Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C<br />
<br />
'''Assembly of the Tris-Glycine gel in the electrophoresis unit'''<br />
*Take the electrophoresis unit of the Hoeffer system and place it next to a power unit<br />
*Dilute 10X and pour 1X electrophoresis buffer in the container( roughly 1 cm high)<br />
*Place gel in the buffer without air bubbles under the gel<br />
*Use the red clamps to place the gel tightly in the unit<br />
*Pour 1X electrophoresis buffer in the chamber so that the top of the gel is immersed<br />
*Take the comb out of the gel and rinse the wells using a hooked needle and syringe<br />
<br />
'''Runninge of the gel'''<br />
*Prepare the samples in 1X SDS sample buffer (NOT nucleic acid loading buffer) <br />
*Boil the samples for 5 min and spin down <br />
*Pipet the samples and protein marker carefully into the wells<br />
*Place the electrode cap on the unit and press lightly.Put the other side of the electrode cables in the correct holes of the power unit<br />
*Switch on the power unit and run until the blue front is appr. 1 cm from the end of the gel. Alternatively, use the prestained protein marker to identify the exact point of stopping<br />
*Disassemble the electrophoresis unit and take the gel<br />
*Take one plate off and cut one corner away for positioning purpose<br />
*Carefully bring the gel into a clean staining container using some demi water and/or spacers<br />
*Stain the gel with CBB or silver</div>Neimahttp://2010.igem.org/Team:Groningen/Protocols_for_Bacillus_subtilis_168Team:Groningen/Protocols for Bacillus subtilis 1682010-09-09T17:22:31Z<p>Neima: New page: '''2-step transformation procedure for ''Bacillus subtilis'' 168''' *Cells are grown O/N at 37 °C in supplemented medium contains Spizizen's salts (without antibiotics) *Dilute c...</p>
<hr />
<div><br />
'''2-step transformation procedure for ''Bacillus subtilis'' 168'''<br />
*Cells are grown O/N at 37 °C in [[supplemented medium]] contains [[Spizizen's salts]] (without antibiotics)<br />
*Dilute cells 10X in fresh medium (10 ml) and grow for 3 h at 37 °C, shaking <br />
*Make agar plates<br />
*Cells are then diluted 1:1 in [[starvation medium]](prewarmed, no antibiotic)<br />
*After 2 hours, approx. 1 μg DNA (BRB689; Cmr ''amyQ+'') is added to 100 μl cells in a 2 ml tube, and the mixture is incubated for 30 min at 37 °C (200 rpm)<br />
*Dilute 3-fold in prewarmed [[TY medium]] and plate after another 45 min (37 °C;200 rpm)<br />
*Dilutions (in starvation medium) for plating are:<br />
* 10^0 and 10^-1 on selective media<br />
* 10^-5 on non-selective plates (viable count)</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-09-09T17:20:54Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Competent Cells Preparation | Competent Cells Preparation]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]<br />
<br />
[[Team:Groningen/Chaplins | Chaplins]]<br />
<br />
[[Team:Groningen/Protocols for Streptomyces | Protocols for Streptomyces]]<br />
<br />
[[Team:Groningen/Antibiotics Concentrationas and Organisms | Antibiotics Concentrations and Organism]]</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-09-09T17:20:27Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Competent Cells Preparation | Competent Cells Preparation]]<br />
<br />
[[Team:Groningen/Growing of Streptomyces | Growing of Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]<br />
<br />
[[Team:Groningen/Chaplins | Chaplins]]<br />
<br />
[[Team:Groningen/Protocols for Streptomyces | Protocols for Streptomyces]]<br />
<br />
[[Team:Groningen/Antibiotics Concentrationas and Organisms | Antibiotics Concentrations and Organism]]</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-09-09T17:19:43Z<p>Neima: </p>
<hr />
<div><br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/Antibiotics_Concentrationas_and_OrganismsTeam:Groningen/Antibiotics Concentrationas and Organisms2010-09-09T17:19:09Z<p>Neima: New page: '''Concentrations for antibiotics ''' '''Antibiotic''' ''' Stock''' ''' E.coli ''' ''' B.subtilis''' *Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C...</p>
<hr />
<div>'''Concentrations for antibiotics ''' <br />
<br />
'''Antibiotic''' ''' Stock''' ''' E.coli ''' ''' B.subtilis'''<br />
<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
<br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
<br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)<br />
<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-09-09T17:15:35Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Competent Cells Preparation | Competent Cells Preparation]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]<br />
<br />
[[Team:Groningen/Chaplins | Chaplins]]<br />
<br />
[[Team:Groningen/Protocols for Streptomyces | Protocols for Streptomyces]]<br />
<br />
[[Team:Groningen/Antibiotics Concentrationas and Organisms | Antibiotics Concentrations and Organism]]</div>Neimahttp://2010.igem.org/Team:Groningen/Protocols_for_StreptomycesTeam:Groningen/Protocols for Streptomyces2010-09-09T17:13:05Z<p>Neima: New page: '''Growth condition''' -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...</p>
<hr />
<div>'''Growth condition'''<br />
<br />
-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
<br />
*Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
<br />
*Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
<br />
*Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
<br />
*Press the loading cilinder on the tower tightl (don't be to careful)<br />
<br />
*Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
<br />
*Put on the nozzle and the lit of the nozzle, tightly<br />
<br />
*The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
<br />
*Adjust your pressure to 13 kpsi<br />
<br />
*Use the pin to start the machine, after two loud klblam!'s remove it<br />
<br />
*Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
<br />
*Run it through another 5X <br />
<br />
*Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
<br />
*Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
-Spin off, 4000 rpm, 5 minutes: Use pellet<br />
<br />
-Cook in 2% SDS<br />
<br />
-Spin off,4000 rpm, 5 min: Use pellet<br />
-Wash 10X with demi-water:<br />
<br />
*Resuspend <br />
*Spin off: Use pellet<br />
*Etc.<br />
<br />
-Freeze pellet (For freeze drying: free it well)<br />
<br />
-Freeze dry your pellet<br />
*Freeze the jar in which you will freeze dry your sample as well<br />
*Turn on the machine, it needs to get on temperature and suck vacuum<br />
<br />
Make sure all the valves are closed<br />
The big red valve needs to be open<br />
The machine is ready when temperature reaches -80 and stays stable<br />
<br />
*Your greiner tube will be put in the jar with either a loose tube hood or parafilm to seal it off (prick 4 holes), seal the jar of with the rubber top<br />
<br />
*Place the jar on one of the valves and open it up slowly<br />
<br />
*When the sample is dry-freezed<br />
<br />
Close the valve and remove the jar<br />
Turn off the machine<br />
Close the red valve</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-09-09T17:11:37Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Competent Cells Preparation | Competent Cells Preparation]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]<br />
<br />
[[Team:Groningen/Chaplins | Chaplins]]<br />
<br />
[[Team:Groningen/Protocols for Streptomyces | Protocols for Streptomyces]]</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-09-09T17:09:40Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
<br />
*Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
<br />
*Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
<br />
*Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
<br />
*Press the loading cilinder on the tower tightl (don't be to careful)<br />
<br />
*Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
<br />
*Put on the nozzle and the lit of the nozzle, tightly<br />
<br />
*The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
<br />
*Adjust your pressure to 13 kpsi<br />
<br />
*Use the pin to start the machine, after two loud klblam!'s remove it<br />
<br />
*Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
<br />
*Run it through another 5X <br />
<br />
*Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
<br />
*Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
-Spin off, 4000 rpm, 5 minutes: Use pellet<br />
<br />
-Cook in 2% SDS<br />
<br />
-Spin off,4000 rpm, 5 min: Use pellet<br />
-Wash 10X with demi-water:<br />
<br />
*Resuspend <br />
*Spin off: Use pellet<br />
*Etc.<br />
<br />
-Freeze pellet (For freeze drying: free it well)<br />
<br />
-Freeze dry your pellet<br />
*Freeze the jar in which you will freeze dry your sample as well<br />
*Turn on the machine, it needs to get on temperature and suck vacuum<br />
<br />
Make sure all the valves are closed<br />
The big red valve needs to be open<br />
The machine is ready when temperature reaches -80 and stays stable<br />
<br />
*Your greiner tube will be put in the jar with either a loose tube hood or parafilm to seal it off (prick 4 holes), seal the jar of with the rubber top<br />
<br />
*Place the jar on one of the valves and open it up slowly<br />
<br />
*When the sample is dry-freezed<br />
<br />
Close the valve and remove the jar<br />
Turn off the machine<br />
Close the red valve<br />
<br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-09-09T17:04:03Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
<br />
*Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
<br />
*Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
<br />
*Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
<br />
*Press the loading cilinder on the tower tightl (don't be to careful)<br />
<br />
*Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
<br />
*Put on the nozzle and the lit of the nozzle, tightly<br />
<br />
*The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
<br />
*Adjust your pressure to 13 kpsi<br />
<br />
*Use the pin to start the machine, after two loud klblam!'s remove it<br />
<br />
*Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
<br />
*Run it through another 5X <br />
<br />
*Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
<br />
*Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
-Spin off, 4000 rpm, 5 minutes: Use pellet<br />
<br />
-Cook in 2% SDS<br />
<br />
-Spin off,4000 rpm, 5 min: Use pellet<br />
-Wash 10X with demi-water:<br />
<br />
*Resuspend <br />
*Spin off: Use pellet<br />
*Etc.<br />
<br />
-Freeze pellet (For freeze drying: free it well)<br />
<br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/ChaplinsTeam:Groningen/Chaplins2010-09-09T17:02:34Z<p>Neima: </p>
<hr />
<div>'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Chaplin purification'''<br />
<br />
-5mg cell wall in 1.5 mL tube. Do not use gloves because static electric interaction<br />
<br />
-Add app. 1 mL of TFA with a glass pasteur pipette.Do this in fumer and never leave TFA open for more than 30 sec!<br />
<br />
-Vortex 1 min<br />
<br />
-Spin down insoluble material for 10 min at 13000 rpm<br />
<br />
-Transfer supernatant to new 1.5 mL eppendorf tube or 5 mL Greiner tube<br />
<br />
-Dry TFA for 1-2 hours<br />
<br />
-When tube is dry, resolve in water or Tris buffer<br />
<br />
'''Sample treatment'''<br />
<br />
-Take 2ml sample and spin down<br />
<br />
'''Supernatant'''<br />
<br />
-1.5 ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)<br />
<br />
-TCA precipitation until drying pellet after washing with aceton<br />
<br />
-Continue with TFA treatment <br />
<br />
'''Cell pellet'''<br />
<br />
-TFA treatment<br />
<br />
-Pellet of TFA treatment with cells should be airdried<br />
<br />
-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)<br />
<br />
-Speedvacum<br />
<br />
-TFA treatment</div>Neimahttp://2010.igem.org/Team:Groningen/ChaplinsTeam:Groningen/Chaplins2010-09-09T14:59:55Z<p>Neima: New page: '''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 (wi...</p>
<hr />
<div>'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Chaplin purification'''<br />
<br />
-5mg cell wall in 1.5 mL tube. Do not use gloves because static electric interaction<br />
<br />
-Add app. 1 mL of TFA with a glass pasteur pipette.Do this in fumer and never leave TFA open for more than 30 sec!<br />
<br />
-Vortex 1 min<br />
<br />
-Spin down insoluble material for 10 min at 13000 rpm<br />
<br />
-Transfer supernatant to new 1.5 mL eppendorf tube or 5 mL Greiner tube<br />
<br />
-Dry TFA for 1-2 hours<br />
<br />
-When tube is dry, resolve in water or Tris buffer</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-09-09T14:57:45Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Competent Cells Preparation | Competent Cells Preparation]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]<br />
<br />
[[Team:Groningen/Chaplins | Chaplins]]</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-09-09T14:55:56Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/Competent Cells Preparation | Competent Cells Preparation]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Protocols for Bacillus subtilis 168 | Protocols for Bacillus subtilis 168]]</div>Neimahttp://2010.igem.org/Procedures_for_Bacillus_subtilis_168Procedures for Bacillus subtilis 1682010-09-09T14:51:44Z<p>Neima: New page: '''2-step transformation procedure for ''Bacillus subtilis'' 168''' *Cells are grown O/N at 37 °C in supplemented medium contains Spizizen's salts (without antibiotics) *Dilute ce...</p>
<hr />
<div>'''2-step transformation procedure for ''Bacillus subtilis'' 168'''<br />
*Cells are grown O/N at 37 °C in [[supplemented medium]] contains [[Spizizen's salts]] (without antibiotics)<br />
*Dilute cells 10X in fresh medium (10 ml) and grow for 3 h at 37 °C, shaking <br />
*Make agar plates<br />
*Cells are then diluted 1:1 in [[starvation medium]](prewarmed, no antibiotic)<br />
*After 2 hours, approx. 1 μg DNA (BRB689; Cmr ''amyQ+'') is added to 100 μl cells in a 2 ml tube, and the mixture is incubated for 30 min at 37 °C (200 rpm)<br />
*Dilute 3-fold in prewarmed [[TY medium]] and plate after another 45 min (37 °C;200 rpm)<br />
*Dilutions (in starvation medium) for plating are:<br />
* 10^0 and 10^-1 on selective media<br />
* 10^-5 on non-selective plates (viable count)</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-09-09T14:49:21Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/CaCl2 Competent Cells |CaCl2 Competent Cells]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Procedures for Bacillus subtilis 168]]</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-08-31T18:07:08Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
<br />
*Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
<br />
*Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
<br />
*Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
<br />
*Press the loading cilinder on the tower tightl (don't be to careful)<br />
<br />
*Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
<br />
*Put on the nozzle and the lit of the nozzle, tightly<br />
<br />
*The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
<br />
*Adjust your pressure to 13 kpsi<br />
<br />
*Use the pin to start the machine, after two loud klblam!'s remove it<br />
<br />
*Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
<br />
*Run it through another 5X <br />
<br />
*Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
<br />
*Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
-Spin off, 4000 rpm, 5 minutes: Use pellet<br />
<br />
-Cook in 2% SDS<br />
<br />
-Spin off,4000 rpm, 5 min: Use pellet<br />
-Wash 10X with demi-water:<br />
<br />
*Resuspend <br />
*Spin off: Use pellet<br />
*Etc.<br />
<br />
-Freeze pellet (For freeze drying: free it well)<br />
<br />
'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Chaplin purification'''<br />
<br />
-5mg cell wall in 1.5 mL tube. Do not use gloves because static electric interaction<br />
<br />
-Add app. 1 mL of TFA with a glass pasteur pipette.Do this in fumer and never leave TFA open for more than 30 sec!<br />
<br />
-Vortex 1 min<br />
<br />
-Spin down insoluble material for 10 min at 13000 rpm<br />
<br />
-Transfer supernatant to new 1.5 mL eppendorf tube or 5 mL Greiner tube<br />
<br />
-Dry TFA for 1-2 hours<br />
<br />
-When tube is dry resolve in water or Tris buffer <br />
<br />
'''Sample treatment'''<br />
<br />
-Take 2ml sample and spin down<br />
'''Supernatant'''<br />
-1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)<br />
<br />
-Do TCA precipitation until drying pellet after washing with aceton<br />
<br />
-Continue with TFA treatment <br />
<br />
'''Cell pellet'''<br />
<br />
-TFA treatment<br />
<br />
-Pellet of TFA treatment with cells should be airdried<br />
<br />
-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)<br />
<br />
-Speedvacum<br />
<br />
-TFA treatment<br />
<br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-08-31T17:37:18Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
<br />
*Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
<br />
*Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
<br />
*Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
<br />
*Press the loading cilinder on the tower tightl (don't be to careful)<br />
<br />
*Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
<br />
*Put on the nozzle and the lit of the nozzle, tightly<br />
<br />
*The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
<br />
*Adjust your pressure to 13 kpsi<br />
<br />
*Use the pin to start the machine, after two loud klblam!'s remove it<br />
<br />
*Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
<br />
*Run it through another 5X <br />
<br />
*Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
<br />
*Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
-Spin off, 4000 rpm, 5 minutes: Use pellet<br />
<br />
-Cook in 2% SDS<br />
<br />
-Spin off,4000 rpm, 5 min: Use pellet<br />
-Wash 10X with demi-water:<br />
<br />
*Resuspend <br />
*Spin off: Use pellet<br />
*Etc.<br />
<br />
-Freeze pellet (For freeze drying: free it well)<br />
<br />
'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Sample treatment'''<br />
<br />
-Take 2ml sample and spin down<br />
'''Supernatant'''<br />
-1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)<br />
<br />
-Do TCA precipitation until drying pellet after washing with aceton<br />
<br />
-Continue with TFA treatment <br />
<br />
'''Cell pellet'''<br />
<br />
-TFA treatment<br />
<br />
-Pellet of TFA treatment with cells should be airdried<br />
<br />
-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)<br />
<br />
-Speedvacum<br />
<br />
-TFA treatment<br />
<br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-08-31T16:47:36Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
<br />
*Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
<br />
*Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
<br />
*Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
<br />
*Press the loading cilinder on the tower tightl (don't be to careful)<br />
<br />
*Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
<br />
*Put on the nozzle and the lit of the nozzle, tightly<br />
<br />
*The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
<br />
*Adjust your pressure to 13 kpsi<br />
<br />
*Use the pin to start the machine, after two loud klblam!'s remove it<br />
<br />
*Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
<br />
*Run it through another 5X <br />
<br />
*Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
<br />
*Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
-Spin off, 4000 rpm, 5 minutes: Use pellet<br />
<br />
-Cook in 2% SDS<br />
<br />
-Spin off,4000 rpm, 5 min: Use pellet<br />
<br />
-Wash 10X with demi-water:<br />
*Resuspend <br />
<br />
*Spin off: Use pellet<br />
<br />
*Etc.<br />
<br />
'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Sample treatment'''<br />
<br />
-Take 2ml sample and spin down<br />
'''Supernatant'''<br />
-1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)<br />
<br />
-Do TCA precipitation until drying pellet after washing with aceton<br />
<br />
-Continue with TFA treatment <br />
<br />
'''Cell pellet'''<br />
<br />
-TFA treatment<br />
<br />
-Pellet of TFA treatment with cells should be airdried<br />
<br />
-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)<br />
<br />
-Speedvacum<br />
<br />
-TFA treatment<br />
<br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-08-28T19:11:06Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
'''Cell wall isolation'''<br />
<br />
-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.<br />
<br />
-Run this suspension throught the cell disrupter at 13 kpsi 6X:<br />
a.Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.<br />
b.Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).<br />
c.Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.<br />
d.Press the loading cilinder on the tower tightl (don't be to careful)<br />
e.Load '''no more than 15 mL''' of your '''filtered''' suspension<br />
f.Put on the nozzle and the lit of the nozzle, tightly<br />
g.The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press<br />
h.Adjust your pressure to 13 kpsi<br />
i.Use the pin to start the machine, after two loud klblam!'s remove it<br />
j.Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL<br />
k.Run it through another 5X<br />
l.Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice<br />
m.Clean all parts of the french style cell distrupter with 70% ethanol<br />
<br />
-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)<br />
<br />
<br />
<br />
'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Sample treatment'''<br />
<br />
-Take 2ml sample and spin down<br />
'''Supernatant'''<br />
-1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)<br />
<br />
-Do TCA precipitation until drying pellet after washing with aceton<br />
<br />
-Continue with TFA treatment <br />
<br />
'''Cell pellet'''<br />
<br />
-TFA treatment<br />
<br />
-Pellet of TFA treatment with cells should be airdried<br />
<br />
-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)<br />
<br />
-Speedvacum<br />
<br />
-TFA treatment<br />
<br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/2-step_Transformation_Procedure_for_Bacillus_subtilis_168Team:Groningen/2-step Transformation Procedure for Bacillus subtilis 1682010-08-28T17:54:28Z<p>Neima: New page: '''2-step transformation procedure for ''Bacillus subtilis'' 168''' *Cells are grown O/N at 37 °C in supplemented medium contains Spizizen's salts (without antibiotics) *Dilute ce...</p>
<hr />
<div>'''2-step transformation procedure for ''Bacillus subtilis'' 168'''<br />
*Cells are grown O/N at 37 °C in [[supplemented medium]] contains [[Spizizen's salts]] (without antibiotics)<br />
*Dilute cells 10X in fresh medium (10 ml) and grow for 3 h at 37 °C, shaking <br />
*Make agar plates<br />
*Cells are then diluted 1:1 in [[starvation medium]](prewarmed, no antibiotic)<br />
*After 2 hours, approx. 1 μg DNA (BRB689; Cmr ''amyQ+'') is added to 100 μl cells in a 2 ml tube, and the mixture is incubated for 30 min at 37 °C (200 rpm)<br />
*Dilute 3-fold in prewarmed [[TY medium]] and plate after another 45 min (37 °C;200 rpm)<br />
*Dilutions (in starvation medium) for plating are:<br />
* 10^0 and 10^-1 on selective media<br />
* 10^-5 on non-selective plates (viable count)</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-08-28T17:54:15Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/CaCl2 Competent Cells |CaCl2 Competent Cells]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/2-step Transformation Procedure for Bacillus subtilis 168]]</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-08-28T17:53:42Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/CaCl2 Competent Cells |CaCl2 Competent Cells]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/2-step Transformation Procedure for ''Bacillus subtilis'' 168]]</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-08-24T23:48:11Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
<br />
'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Sample treatment'''<br />
<br />
-Take 2ml sample and spin down<br />
'''Supernatant'''<br />
-1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)<br />
<br />
-Do TCA precipitation until drying pellet after washing with aceton<br />
<br />
-Continue with TFA treatment <br />
<br />
'''Cell pellet'''<br />
<br />
-TFA treatment<br />
<br />
-Pellet of TFA treatment with cells should be airdried<br />
<br />
-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)<br />
<br />
-Speedvacum<br />
<br />
-TFA treatment<br />
<br />
'''Concentrations for antibiotics ''' <br />
<br />
Antibiotic Stock E.coli B.subtilis<br />
*Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500) <br />
*Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)<br />
*Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)<br />
*Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6) <br />
*Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)<br />
*Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)<br />
*Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)<br />
*Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*<br />
*Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)</div>Neimahttp://2010.igem.org/Team:Groningen/Growing_StreptomycesTeam:Groningen/Growing Streptomyces2010-08-24T23:02:50Z<p>Neima: </p>
<hr />
<div>-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 <br />
<br />
*Agar 6 g <br />
*D-mannitol 6 g <br />
*Soya flour 6 g <br />
*Demiwater 300ml <br />
*Plating ΔrdeAB spores 10^5 /plate <br />
<br />
-Incubate for 7 days at 30 °C <br />
<br />
-Harvest mycelium with razorblade<br />
<br />
<br />
'''Expression Chaplins in NZ8900'''<br />
<br />
-Grow O/N culture in Ty with antibiotics (Km5 and Cm5)<br />
<br />
-Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)<br />
<br />
-Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1<br />
<br />
-Grow till OD600 is approx. 0.5<br />
<br />
-Take 2 ml t=0 sample<br />
<br />
-Induce with 1% subtiline<br />
<br />
-Continue growing<br />
<br />
-Take 2 ml samples every hour<br />
<br />
'''Sample treatment'''<br />
<br />
-Take 2ml sample and spin down<br />
'''Supernatant'''<br />
-1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)<br />
<br />
-Do TCA precipitation until drying pellet after washing with aceton<br />
<br />
-Continue with TFA treatment <br />
<br />
'''Cell pellet'''<br />
<br />
-TFA treatment<br />
<br />
-Pellet of TFA treatment with cells should be airdried<br />
<br />
-Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)<br />
<br />
-Speedvacum<br />
<br />
-TFA treatment</div>Neimahttp://2010.igem.org/Team:Groningen/CaCl2_Competent_CellsTeam:Groningen/CaCl2 Competent Cells2010-08-24T22:44:09Z<p>Neima: </p>
<hr />
<div>'''Day 1:''' <br />
<br />
-Inoculate 5 ml [[LB( TY) medium]] with ''E.coli'' from glycerol stock <br />
<br />
-Grow overnight at 37 ºC <br />
<br />
'''Day 2:''' <br />
<br />
-Inoculate 20 ml LB medium with 200 μl overnight culture <br />
<br />
-Grow at 37 ºC until OD600 = 0,3-0,5(+/- 2 hours) <br />
<br />
-Spin down 5 minute at 4000 rpm at 4 ºC <br />
<br />
-Resuspend in 10 ml chilled 0,1 M CaCl2 (from here, keep on ice! ) <br />
<br />
-Inoculate on ice for 20 minutes <br />
<br />
-Spin down as before <br />
<br />
-Remove supernatant <br />
<br />
-Resuspend in 2 ml 0,1 M CaCl2 /10% glycerol <br />
<br />
-Divide 100 μl aliquots <br />
<br />
-Store competent cells in - 80 ºC<br />
<br />
'''Transformation protocol'''<br />
<br />
Melt agar in autoclave or microwave. Cool down to 60 ºC. One jar ( 100mL ) is enough for 7 plates.<br />
<br />
-Add 10 μl of ligation product to 100 μl of cells ( don't for get controls! )<br />
<br />
-Incubate 30 min on ice (make agar plates)<br />
<br />
-Incubate for 1 min at 42 ºC <br />
<br />
-Incubate cells on ice for 2 min<br />
<br />
-Add 400 μl of [[SOC medium]]<br />
<br />
-Incubate for 30 min at 37 ºC on shaker<br />
<br />
-Spread 100-300 μl onto a plate made with appropriate antibiotic<br />
<br />
-Grow overnight at 37 °C. <br />
<br />
-Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.<br />
<br />
*If you expect the transformation to be very efficient (for instance using undigested plasmid DNA) first make dilution (1:10 or 1:100 ) and plate 250 μl of this<br />
<br />
*If you expect the trasformation to be inefficient, concentrate cells by spinning the 500 μl and resuspending the pellet in 250 μl, which you will use for plating.<br />
<br />
'''Glycerol stock'''<br />
<br />
Materials<br />
<br />
*40% glycerol solution<br />
*Cryogenic vials<br />
<br />
Methods<br />
<br />
*Add 1 ml of 40% glycerol in H2O to a cryogenic vial<br />
*Add 1 ml sample from the culture of the bacteria to be stored( or,take 1 ml culture and add 300 μl of the 87% glycerol)<br />
*Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed<br />
*Alternatively, pipet to mix<br />
*Use a tough spot to put the name of the strain or some useful identifier on the top of the vial<br />
*On the side of the vial list all relavant information-part,vector,strain, date,researcher,etc<br />
*Store in a freezer box in a -80 ºC freezer.Remember to record where vial is stored for fast retrieval later<br />
<br />
Note: Ideally, your culture should be in logarithmic growth phase</div>Neimahttp://2010.igem.org/TY_mediumTY medium2010-08-24T22:32:31Z<p>Neima: New page: '''Ingredients''' -10 g Bacto-tryptone -5 g yeast extract -10 g NaCl '''Protocol''' -Mix dry ingredients and add distilled water up to 1 Liet -Pour into 2 L flask (or greater) -Auto...</p>
<hr />
<div>'''Ingredients'''<br />
<br />
-10 g Bacto-tryptone<br />
<br />
-5 g yeast extract<br />
<br />
-10 g NaCl<br />
<br />
'''Protocol'''<br />
<br />
-Mix dry ingredients and add distilled water up to 1 Liet<br />
<br />
-Pour into 2 L flask (or greater)<br />
<br />
-Autoclave (liquid cycle)<br />
<br />
(250 oF,22 psi, 30 min)<br />
<br />
''Note:'' <br />
There asre two formulations of LB, Miller and Lennox, that differ in the amount of NaCl. Lenox has less salt, only 5 g/L. The Qiagen miniprep kit recommends LB with 10 g NaCl for highest plasmid yields.<br />
<br />
''Notes''<br />
We don't adjust pH of medium when we make it on the fly. However, if it is really important, pH the medium to 7.0 with 5M NaOH (~ 200 μl ).</div>Neimahttp://2010.igem.org/Starvation_mediumStarvation medium2010-08-24T22:30:07Z<p>Neima: New page: Starvation medium: 50 ml 1X Spizizen's salts+ 0.5% glucose (better not to add antibiotics)</p>
<hr />
<div>Starvation medium: 50 ml 1X Spizizen's salts+ 0.5% glucose (better not to add antibiotics)</div>Neimahttp://2010.igem.org/Supplemented_mediumSupplemented medium2010-08-24T22:28:10Z<p>Neima: New page: *50 ml 1X diluted '''Spizizen's salts''' *0.5 ml(0.02%) '''casamino acids''' (2%) *0.5 ml(14 µg/ml) '''tryptophane''' (100X) *1.25 ml (0.5%)'''glucose''' (20%) *50 µl (22 µg/ml) '''FeN...</p>
<hr />
<div>*50 ml 1X diluted '''Spizizen's salts'''<br />
*0.5 ml(0.02%) '''casamino acids''' (2%) <br />
*0.5 ml(14 µg/ml) '''tryptophane''' (100X)<br />
*1.25 ml (0.5%)'''glucose''' (20%)<br />
*50 µl (22 µg/ml) '''FeNH4 citrate''' (1000X) (prepare fresh every 2 weeks! )<br />
*antibiotics</div>Neimahttp://2010.igem.org/Spizizen%27s_saltsSpizizen's salts2010-08-24T20:12:17Z<p>Neima: New page: Spizizen's 5X diluted salts solution contains of (per liter): *K2HPO4, 14.8 g/l (85 mM) *KH2PO4, 5.4 g/l (40 mM) *(NH4)2SO4, ...</p>
<hr />
<div>Spizizen's 5X diluted salts solution contains of (per liter):<br />
<br />
*K2HPO4, 14.8 g/l (85 mM)<br />
<br />
*KH2PO4, 5.4 g/l (40 mM)<br />
<br />
*(NH4)2SO4, 2 g/l (15 mM)<br />
<br />
*''tri''-Sodium citrate.2H2O, 1.9 g/l (6 mM)<br />
<br />
*MgSO4.7H2O, 0.2 g/l (0.8mM)<br />
<br />
pH7.0; adjusted with 10 N NaOH</div>Neimahttp://2010.igem.org/Team:Groningen/Normal_SDS-PAGE_using_Tris-Glycine_Gels_and_Electrophoresis_BufferTeam:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer2010-08-24T20:03:34Z<p>Neima: </p>
<hr />
<div>'''Wear gloves at all times!''' <br />
<br />
Requirements:<br />
*30% acrylamide(Biorad) '''NEUROTOXIN!'''<br />
*4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8<br />
*4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol<br />
*TEMED( in yellow cabinet )<br />
*10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge<br />
<br />
'''Hoeffer Mighty gel system'''<br />
Casting of the gel:<br />
*Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)<br />
*Clean the plates etc with soap, rinse with demiwater and ethanol, and dry<br />
*Assemble the system in the gel casting holder. Mark the line of separation/stacking<br />
*Mix the separation gel in 10 ml plastic tube:<br />
'''for 2 gels'''<br />
'''Seperation gel (0.75 mm)''' ''' 12.5%''' '''16%''' <br />
30% acrylamide 4 ml 5.1 ml<br />
4X separation buffer 2.4 ml 2.4 ml<br />
MQ 3.2 ml 2.1 ml<br />
10% APS 28 μl 28 μl<br />
TEMED 28 μl 28 μl<br />
*Pipet the separation gel mix immediately in between the glass plates until the marked line is reached<br />
*Pipet water-saturated isobutanol on top of the polymerizing separation gel<br />
*Let the separation gel polymerize completely before preparing the stacking gel<br />
*When the gel is polymerized, discard the isobutanol and wash the gel with water<br />
*Mix the stacking gel in a 10 ml tube:<br />
'''for 2 gels'''<br />
mQ 1.92 ml<br />
4X stacking buffer 0.83 ml<br />
30% acrylamide 560 μl<br />
10% APS 14 μl<br />
TEMED 7 μl<br />
*Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker<br />
*Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube<br />
*Disassemble the gel casting holder and take out the gel/plates<br />
*Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C<br />
<br />
'''Assembly of the Tris-Glycine gel in the electrophoresis unit'''<br />
*Take the electrophoresis unit of the Hoeffer system and place it next to a power unit<br />
*Dilute 10X and pour 1X electrophoresis buffer in the container( roughly 1 cm high)<br />
*Place gel in the buffer without air bubbles under the gel<br />
*Use the red clamps to place the gel tightly in the unit<br />
*Pour 1X electrophoresis buffer in the chamber so that the top of the gel is immersed<br />
*Take the comb out of the gel and rinse the wells using a hooked needle and syringe<br />
<br />
'''Runninge of the gel'''<br />
*Prepare the samples in 1X SDS sample buffer (NOT nucleic acid loading buffer) <br />
*Boil the samples for 5 min and spin down <br />
*Pipet the samples and protein marker carefully into the wells<br />
*Place the electrode cap on the unit and press lightly.Put the other side of the electrode cables in the correct holes of the power unit<br />
*Switch on the power unit and run until the blue front is appr. 1 cm from the end of the gel. Alternatively, use the prestained protein marker to identify the exact point of stopping<br />
*Disassemble the electrophoresis unit and take the gel<br />
*Take one plate off and cut one corner away for positioning purpose<br />
*Carefully bring the gel into a clean staining container using some demi water and/or spacers<br />
*Stain the gel with CBB or silver<br />
<br />
<br />
'''2-step transformation procedure for ''Bacillus subtilis'' 168'''<br />
*Cells are grown O/N at 37 °C in [[supplemented medium]] contains [[Spizizen's salts]] (without antibiotics)<br />
*Dilute cells 10X in fresh medium (10 ml) and grow for 3 h at 37 °C, shaking <br />
*Make agar plates<br />
*Cells are then diluted 1:1 in [[starvation medium]](prewarmed, no antibiotic)<br />
*After 2 hours, approx. 1 μg DNA (BRB689; Cmr ''amyQ+'') is added to 100 μl cells in a 2 ml tube, and the mixture is incubated for 30 min at 37 °C (200 rpm)<br />
*Dilute 3-fold in prewarmed [[TY medium]] and plate after another 45 min (37 °C;200 rpm)<br />
*Dilutions (in starvation medium) for plating are:<br />
* 10^0 and 10^-1 on selective media<br />
* 10^-5 on non-selective plates (viable count)</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-08-24T19:46:25Z<p>Neima: /* Protocols */</p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/CaCl2 Competent Cells |CaCl2 Competent Cells]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-08-24T19:44:48Z<p>Neima: </p>
<hr />
<div>== Protocols == <br />
<br />
[[Team:Groningen/CaCl2 Competent Cells |CaCl2 Competent Cells]]<br />
<br />
[[Team:Groningen/Growing Streptomyces | Growing Streptomyces]]<br />
<br />
[[Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer | Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]<br />
<br />
[[Team:Groningen/Two step transformation procedure for ''Bacillus subtilis''168&8G5 ]]</div>Neimahttp://2010.igem.org/Normal_SDS-PAGE_using_Tris-Glycine_Gels_and_Electrophoresis_BufferNormal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer2010-08-24T13:58:13Z<p>Neima: </p>
<hr />
<div>'''Wear gloves at all times!''' <br />
<br />
Requirements:<br />
*30% acrylamide(Biorad) '''NEUROTOXIN!'''<br />
*4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8<br />
*4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol<br />
*TEMED( in yellow cabinet )<br />
*10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge<br />
<br />
'''Hoeffer Mighty gel system'''<br />
Casting of the gel:<br />
*Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)<br />
*Clean the plates etc with soap, rinse with demiwater and ethanol, and dry<br />
*Assemble the system in the gel casting holder. Mark the line of separation/stacking<br />
*Mix the separation gel in 10 ml plastic tube:<br />
'''for 2 gels'''<br />
'''Seperation gel (0.75 mm)''' ''' 12.5%''' '''16%''' <br />
30% acrylamide 4 ml 5.1 ml<br />
4X separation buffer 2.4 ml 2.4 ml<br />
MQ 3.2 ml 2.1 ml<br />
10% APS 28 μl 28 μl<br />
TEMED 28 μl 28 μl<br />
*Pipet the separation gel mix immediately in between the glass plates until the marked line is reached<br />
*Pipet water-saturated isobutanol on top of the polymerizing separation gel<br />
*Let the separation gel polymerize completely before preparing the stacking gel<br />
*When the gel is polymerized, discard the isobutanol and wash the gel with water<br />
*Mix the stacking gel in a 10 ml tube:<br />
'''for 2 gels'''<br />
mQ 1.92 ml<br />
4X stacking buffer 0.83 ml<br />
30% acrylamide 560 μl<br />
10% APS 14 μl<br />
TEMED 7 μl<br />
*Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker<br />
*Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube<br />
*Disassemble the gel casting holder and take out the gel/plates<br />
*Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C<br />
<br />
'''Assembly of the Tris-Glycine gel in the electrophoresis unit'''<br />
*Take the electrophoresis unit of the Hoeffer system and place it next to a power unit<br />
*Dilute 10X and pour 1X electrophoresis buffer in the container( roughly 1 cm high)<br />
*Place gel in the buffer without air bubbles under the gel<br />
*Use the red clamps to place the gel tightly in the unit<br />
*Pour 1X electrophoresis buffer in the chamber so that the top of the gel is immersed<br />
*Take the comb out of the gel and rinse the wells using a hooked needle and syringe<br />
<br />
'''Runninge of the gel'''<br />
*Prepare the samples in 1X SDS sample buffer (NOT nucleic acid loading buffer) <br />
*Boil the samples for 5 min and spin down <br />
*Pipet the samples and protein marker carefully into the wells<br />
*Place the electrode cap on the unit and press lightly.Put the other side of the electrode cables in the correct holes of the power unit<br />
*Switch on the power unit and run until the blue front is appr. 1 cm from the end of the gel. Alternatively, use the prestained protein marker to identify the exact point of stopping<br />
*Disassemble the electrophoresis unit and take the gel<br />
*Take one plate off and cut one corner away for positioning purpose<br />
*Carefully bring the gel into a clean staining container using some demi water and/or spacers<br />
*Stain the gel with CBB or silver</div>Neimahttp://2010.igem.org/Normal_SDS-PAGE_using_Tris-Glycine_Gels_and_Electrophoresis_BufferNormal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer2010-08-24T13:48:26Z<p>Neima: </p>
<hr />
<div>'''Wear gloves at all times!''' <br />
<br />
Requirements:<br />
*30% acrylamide(Biorad) '''NEUROTOXIN!'''<br />
*4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8<br />
*4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol<br />
*TEMED( in yellow cabinet )<br />
*10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge<br />
<br />
'''Hoeffer Mighty gel system'''<br />
Casting of the gel:<br />
*Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)<br />
*Clean the plates etc with soap, rinse with demiwater and ethanol, and dry<br />
*Assemble the system in the gel casting holder. Mark the line of separation/stacking<br />
*Mix the separation gel in 10 ml plastic tube:<br />
'''for 2 gels'''<br />
'''Seperation gel (0.75 mm)''' ''' 12.5%''' '''16%''' <br />
30% acrylamide 4 ml 5.1 ml<br />
4X separation buffer 2.4 ml 2.4 ml<br />
MQ 3.2 ml 2.1 ml<br />
10% APS 28 μl 28 μl<br />
TEMED 28 μl 28 μl<br />
*Pipet the separation gel mix immediately in between the glass plates until the marked line is reached<br />
*Pipet water-saturated isobutanol on top of the polymerizing separation gel<br />
*Let the separation gel polymerize completely before preparing the stacking gel<br />
*When the gel is polymerized, discard the isobutanol and wash the gel with water<br />
*Mix the stacking gel in a 10 ml tube:<br />
'''for 2 gels'''<br />
mQ 1.92 ml<br />
4X stacking buffer 0.83 ml<br />
30% acrylamide 560 μl<br />
10% APS 14 μl<br />
TEMED 7 μl<br />
*Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker<br />
*Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube<br />
*Disassemble the gel casting holder and take out the gel/plates<br />
*Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C<br />
<br />
'''Assembly of the Tris-Glycine gel in the electrophoresis unit'''</div>Neimahttp://2010.igem.org/Normal_SDS-PAGE_using_Tris-Glycine_Gels_and_Electrophoresis_BufferNormal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer2010-08-24T13:26:29Z<p>Neima: New page: '''Wear gloves at all times!''' Requirements: *30% acrylamide(Biorad) '''NEUROTOXIN!''' *4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8 *4X stacking gel buffer: 0.5M Tris.HCl, 1...</p>
<hr />
<div>'''Wear gloves at all times!''' <br />
<br />
Requirements:<br />
*30% acrylamide(Biorad) '''NEUROTOXIN!'''<br />
*4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8<br />
*4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol<br />
*TEMED( in yellow cabinet )<br />
*10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge<br />
<br />
Hoeffer Mighty gel system</div>Neimahttp://2010.igem.org/Team:Groningen/ProtocolsTeam:Groningen/Protocols2010-08-24T13:22:55Z<p>Neima: </p>
<hr />
<div>== Protocols == <br />
<br />
[[CaCl2 Competent Cells]]<br />
<br />
[[Growing Streptomyces]]<br />
<br />
[[Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer]]</div>Neima