http://2009.igem.org/wiki/index.php?title=Special:Contributions/TOHRU&feed=atom&limit=50&target=TOHRU&year=&month=2009.igem.org - User contributions [en]2024-03-28T21:00:28ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T08:02:35Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Material and Methods=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C<br />
<br />
==Restriction Digestion==<br />
For cloning experiments, the final volume should be 20 or 50<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:29:13Z<p>TOHRU: /* Restriction Digestion */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C<br />
<br />
==Restriction Digestion==<br />
For cloning experiments, the final volume should be 20 or 50<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:28:29Z<p>TOHRU: /* Restriction Digestion */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C<br />
<br />
==Restriction Digestion==<br />
===Cloning experiment===<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:27:19Z<p>TOHRU: /* =For cloning experiment */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C<br />
<br />
==Restriction Digestion==<br />
===For cloning experiment===<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:26:46Z<p>TOHRU: /* Restriction Digestion */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C<br />
<br />
==Restriction Digestion==<br />
===For cloning experiment==<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:25:53Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C<br />
<br />
==Restriction Digestion==<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:25:25Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C<br />
<br />
==Restriction Digestion==</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:24:46Z<p>TOHRU: /* Transformation of chemical competent cell */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
;#Watch the plate and find colony<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:24:12Z<p>TOHRU: /* Plasmid mini prep (QIAprep Spin Miniprep kit) */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:23:46Z<p>TOHRU: /* Plasmid mini prep (QIAprep Spin Miniprep kit) */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!<br />
P1 buffer must be stocked at 4°C<br />
;#Add '''250 ul Buffer P2''' and inventing the tubes 4-6times<br />
;#Add '''350 ul Buffer N3''' and inventing the tubes 4-6times soon after step 5<br />
;#Centrifuge:15krpm, 10min, 4°C<br />
;#Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting<br />
;#Centrifuge: 13krpm, 1min, r.t.<br />
;#Reapply the flow-through to column for improving yield<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''500 ul Buffer PB''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Add '''750 ul Buffer PE''' in column<br />
;#Centrifuge: 13krpm, 1min, r.t. and discard the flow-through<br />
;#Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through<br />
;#Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column<br />
;#Keep at r.t. for 3~5min<br />
;#Centrifuge:15krpm, 1min<br />
;#stock -30°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:07:12Z<p>TOHRU: /* Plasmid mini prep (QIAprep Spin Miniprep kit) */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!<br><br />
P1 solution must be stocked at 4<br />
;#Add '''250 ul Buffer P2'''<br />
<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:05:48Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
<br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!<br />
;#Add<br />
<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T07:05:16Z<p>TOHRU: </p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
</div><br />
<br />
==Plasmid mini prep (QIAprep Spin Miniprep kit)==<br />
;#Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)<br />
;#Incubate overnight at 37°C <br />
;#Harvest the cells by centrifugation 13krpm, 1min, r.t.<br />
;#Resuspend pelleted cells in '''250 ul Buffer P1'''. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!<br />
;#Add</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:28:58Z<p>TOHRU: </p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:26:30Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:26:05Z<p>TOHRU: </p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==<u>Preparing CaCl<sub>2</sub>competent cell</u> ==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==<u>Transformation of chemical competent cell</u>==<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:25:24Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell ==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:24:36Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
===Preparing CaCl<sub>2</sub>competent cell ===<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
===Transformation of chemical competent cell===<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:22:28Z<p>TOHRU: /* Lab protocol */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
=Lab protocol=<br />
<br />
==Preparing CaCl<sub>2</sub>competent cell ==<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
==Transformation of chemical competent cell==<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:21:42Z<p>TOHRU: /* 2009/08/02 */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==Lab protocol==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
====Transformation of chemical competent cell====<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T06:20:49Z<p>TOHRU: /* 2009/08/02 */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==2009/08/02==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
====Transformation of chemical competent cell====<br />
<br />
;#Thaw competent cells on ice<br />
;#Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube<br />
;#On ice for 30min<br />
;#Heat shock 42℃ for 2~1min<br />
;#On ice for 5min<br />
;#Add 900 ul LB<br />
;#Incubate at 37℃ for 1hour (During this, warm plates at 42℃)<br />
;#Harvest cells by centrifuge (14krpm, 1min, r.t.)<br />
;#Discard 900~800 ul of supernatant<br />
;#Resuspend pellet by pipetting<br />
;#Plating all cells on the plate with appropriate antibiotics by glass beads<br />
;#Incubate overnight at 37°C</div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T05:53:53Z<p>TOHRU: /* Preparing CaCl2competent cell */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==2009/08/02==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
;#Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
====Transformationの方法====<br />
<br />
;;;;;#ウェルに15μl milliQ水をいれてピペッティング<br />
;;;;;#エッペンTubeに回収、ナンバリングを忘れないように。(DNAプレートに書いてある数とアルファベット、その上にプレートナンバーを書く。)<br />
;;;;;#コンピ100μlにつき、DNAを2μl加える。<br />
;;;;;#30min On ice<br />
;;;;;#2min,42℃でHeat shock<br />
;;;;;#5min on ice<br />
;;;;;#LBを900μl入れる。<br />
;;;;;#37℃で22min Incubate<br />
;;;;;#platingを行う。プレートにエッペンTubeの内容物をあけ、autoclaveされたビーズをいれてふる。<br />
;;;;;#プレートに日付、名前をかいて、37℃でIncubate。<br />
;;;;;以上です。不明な点はのりさん、とりっぴー、ただしまでどうぞ。文責 中村 匡<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T05:53:22Z<p>TOHRU: /* Preparing CaCl2competent cell */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==2009/08/02==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
;#Pick a single colony (Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
====Transformationの方法====<br />
<br />
;;;;;#ウェルに15μl milliQ水をいれてピペッティング<br />
;;;;;#エッペンTubeに回収、ナンバリングを忘れないように。(DNAプレートに書いてある数とアルファベット、その上にプレートナンバーを書く。)<br />
;;;;;#コンピ100μlにつき、DNAを2μl加える。<br />
;;;;;#30min On ice<br />
;;;;;#2min,42℃でHeat shock<br />
;;;;;#5min on ice<br />
;;;;;#LBを900μl入れる。<br />
;;;;;#37℃で22min Incubate<br />
;;;;;#platingを行う。プレートにエッペンTubeの内容物をあけ、autoclaveされたビーズをいれてふる。<br />
;;;;;#プレートに日付、名前をかいて、37℃でIncubate。<br />
;;;;;以上です。不明な点はのりさん、とりっぴー、ただしまでどうぞ。文責 中村 匡<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T05:53:00Z<p>TOHRU: /* Preparing CaCl2competent cell */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==2009/08/02==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
;#Pick a single colony (strain:Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
====Transformationの方法====<br />
<br />
;;;;;#ウェルに15μl milliQ水をいれてピペッティング<br />
;;;;;#エッペンTubeに回収、ナンバリングを忘れないように。(DNAプレートに書いてある数とアルファベット、その上にプレートナンバーを書く。)<br />
;;;;;#コンピ100μlにつき、DNAを2μl加える。<br />
;;;;;#30min On ice<br />
;;;;;#2min,42℃でHeat shock<br />
;;;;;#5min on ice<br />
;;;;;#LBを900μl入れる。<br />
;;;;;#37℃で22min Incubate<br />
;;;;;#platingを行う。プレートにエッペンTubeの内容物をあけ、autoclaveされたビーズをいれてふる。<br />
;;;;;#プレートに日付、名前をかいて、37℃でIncubate。<br />
;;;;;以上です。不明な点はのりさん、とりっぴー、ただしまでどうぞ。文責 中村 匡<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T05:52:25Z<p>TOHRU: /* 2009/08/02 */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==2009/08/02==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
<b>preparation</b><br />
;#Pick a single colony (strain:Nova Blue) and inoculate 5 ml LB<br />
;#Incubate overnight at 37°C <br />
;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4°C<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M MgCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice for 10min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant<br />
;#Resuspend each pellet in 20 ml chilled '''0.1 M CaCl<sub>2</sub>''' and add further 15 ml 0.1 M MgCl (total 45 ml)<br />
;#On ice 30min<br />
;#Centrifuge:8krpm,5min,4℃<br />
;#Discard supernatant carefuly<br />
;#Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl<sub>2</sub> and 750 µl pre-chilled 50%(v/v) Glycerol<br />
;#Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃<br />
;#stock at -80℃<br />
<br />
<br />
<br />
====Transformationの方法====<br />
<br />
;;;;;#ウェルに15μl milliQ水をいれてピペッティング<br />
;;;;;#エッペンTubeに回収、ナンバリングを忘れないように。(DNAプレートに書いてある数とアルファベット、その上にプレートナンバーを書く。)<br />
;;;;;#コンピ100μlにつき、DNAを2μl加える。<br />
;;;;;#30min On ice<br />
;;;;;#2min,42℃でHeat shock<br />
;;;;;#5min on ice<br />
;;;;;#LBを900μl入れる。<br />
;;;;;#37℃で22min Incubate<br />
;;;;;#platingを行う。プレートにエッペンTubeの内容物をあけ、autoclaveされたビーズをいれてふる。<br />
;;;;;#プレートに日付、名前をかいて、37℃でIncubate。<br />
;;;;;以上です。不明な点はのりさん、とりっぴー、ただしまでどうぞ。文責 中村 匡<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T05:40:58Z<p>TOHRU: /* 2009/08/02 */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==2009/08/02==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
<b>#preparation</b><br />
;;;;;;#Pick a single colony (strain:Nova Blue) and inoculate 5 ml LB<br />
;;;;;;#Incubate overnight at 37°C <br />
;;;;;;#Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer<br />
;;;;;;#Incubate culture at 37°C on a shaker up to OD<sub>600</sub>=0.3~0.5 (Measure OD value first 1hr and each 30min)<br />
;;;;;;#When the culture reaches an OD<sub>600</sub> between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)<br />
;;;;;;#Keep cells on ice for 10min<br />
;;;;;;#Centrifuge:8krpm, 5min,4°C<br />
;;;;;;#Carefully discard supernatant<br />
;;;;;;#0.1M MgCl2溶液(これも使う直前に冷やす)をそれぞれ最初にピペッターで15ml加えてvortex,その後同じものをさらに10ml加える。<br />
;;;;;;#On ice 10min冷蔵室に放置<br />
;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。<br />
;;;;;;#上澄みをデカンテーション。<br />
;;;;;;#0.1M CaCl2(冷やしておく)をピペッターで25ml加えてvortex、さらに10ml加える。<br />
;;;;;;#on ice 30min冷蔵室に放置。<br />
;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。<br />
;;;;;;#上澄みをデカンテーション。(注意深く)<br />
;;;;;;#ピペットマンで750μl 0.1M CaCl2、750μl 50% グリセロール(すべて冷やされているもの)を加えてvortex。<br />
;;;;;;#-80℃に冷やしたペンTubeに遠心分離管の内容物をピペットマンで50μlずつ分注。再び-80℃で保管。<br />
;;;;;;以上です。<br />
<br />
====Transformationの方法====<br />
<br />
;;;;;#ウェルに15μl milliQ水をいれてピペッティング<br />
;;;;;#エッペンTubeに回収、ナンバリングを忘れないように。(DNAプレートに書いてある数とアルファベット、その上にプレートナンバーを書く。)<br />
;;;;;#コンピ100μlにつき、DNAを2μl加える。<br />
;;;;;#30min On ice<br />
;;;;;#2min,42℃でHeat shock<br />
;;;;;#5min on ice<br />
;;;;;#LBを900μl入れる。<br />
;;;;;#37℃で22min Incubate<br />
;;;;;#platingを行う。プレートにエッペンTubeの内容物をあけ、autoclaveされたビーズをいれてふる。<br />
;;;;;#プレートに日付、名前をかいて、37℃でIncubate。<br />
;;;;;以上です。不明な点はのりさん、とりっぴー、ただしまでどうぞ。文責 中村 匡<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/MeetingTeam:Osaka/Meeting2009-08-11T05:21:10Z<p>TOHRU: /* 2009/08/02 */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
From [[Team:Osaka/Notebook|Notebook]]<br />
<br />
==2009/08/02==<br />
<br />
====Preparing CaCl<sub>2</sub>competent cell ====<br />
<b>pre cluture</b><br />
;;;;;;# <br />
;;;;;;# Single colonyを試験管にとり、5mlのLBを加える。<br />
;;;;;;# 37度で一晩おく。<br />
;;;;;;# 大量に菌が得られたら、40mlのLBを入れた三角フラスコ2つに菌をいれて37度で約2時間Incubate。<br />
;;;;;;#分光光度計でOD60=0.3になるまでIncubate。<br />
;;;;;;#OD60=0.3になったら、三角フラスコをOn ice 10min (常に道具は冷やした状態をこころがける)<br />
;;;;;;#遠心分離管(J.20、サイズに注意、冷やしておく)にピペッターで20mlずつ4本分注。はかりで測って重さを均一にする。<br />
;;;;;;#8krpm,4℃、roter ID 20、5min で遠心分離。(ローターのサイズにも注意,20 を使う)<br />
;;;;;;#上澄みをデカンテーション。<br />
;;;;;;#0.1M MgCl2溶液(これも使う直前に冷やす)をそれぞれ最初にピペッターで15ml加えてvortex,その後同じものをさらに10ml加える。<br />
;;;;;;#On ice 10min冷蔵室に放置<br />
;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。<br />
;;;;;;#上澄みをデカンテーション。<br />
;;;;;;#0.1M CaCl2(冷やしておく)をピペッターで25ml加えてvortex、さらに10ml加える。<br />
;;;;;;#on ice 30min冷蔵室に放置。<br />
;;;;;;#8krpm,4℃,roterID 20, 5minで遠心分離。<br />
;;;;;;#上澄みをデカンテーション。(注意深く)<br />
;;;;;;#ピペットマンで750μl 0.1M CaCl2、750μl 50% グリセロール(すべて冷やされているもの)を加えてvortex。<br />
;;;;;;#-80℃に冷やしたペンTubeに遠心分離管の内容物をピペットマンで50μlずつ分注。再び-80℃で保管。<br />
;;;;;;以上です。<br />
<br />
====Transformationの方法====<br />
<br />
;;;;;#ウェルに15μl milliQ水をいれてピペッティング<br />
;;;;;#エッペンTubeに回収、ナンバリングを忘れないように。(DNAプレートに書いてある数とアルファベット、その上にプレートナンバーを書く。)<br />
;;;;;#コンピ100μlにつき、DNAを2μl加える。<br />
;;;;;#30min On ice<br />
;;;;;#2min,42℃でHeat shock<br />
;;;;;#5min on ice<br />
;;;;;#LBを900μl入れる。<br />
;;;;;#37℃で22min Incubate<br />
;;;;;#platingを行う。プレートにエッペンTubeの内容物をあけ、autoclaveされたビーズをいれてふる。<br />
;;;;;#プレートに日付、名前をかいて、37℃でIncubate。<br />
;;;;;以上です。不明な点はのりさん、とりっぴー、ただしまでどうぞ。文責 中村 匡<br />
<br />
</div></div>TOHRUhttp://2009.igem.org/Team:Osaka/NotebookTeam:Osaka/Notebook2009-08-11T04:57:04Z<p>TOHRU: /* Meeting */</p>
<hr />
<div>{{Template:Osaka1}}<br />
<br />
<div style="width: 700px; margin-left: 200px; float:center;"> <br />
<br />
<br />
<table align=center cellpadding=5><br />
<caption><big>'''2009'''</big></caption><br />
<!--<br />
<tr><br />
<th valign=top>{{#calendar: title=February |year=2009 | month=02}}</th><th valign=top>{{#calendar: title=March |year=2009 | month=03}}</th><th valign=top>{{#calendar: title=April |year=2009 | month=04}}</th><th valign=top>{{#calendar: title=May |year=2009 | month=05}}</th><th valign=top>{{#calendar: title=June |year=2009 | month=06}}</th><br />
</tr><br />
--><br />
<tr><br />
<th valign=top>{{#calendar: title=July |year=2009 | month=07}}</th><th valign=top>{{#calendar: title=August |year=2009 | month=08}}</th><th valign=top>{{#calendar: title=September |year=2009 | month=09}}</th><th valign=top>{{#calendar: title=August |year=2009 | month=10}}</th><th valign=top>{{#calendar: title=November |year=2009 | month=11}}</th><br />
</tr><br />
</table><br />
<br />
<br />
==[[Team:Osaka/Meeting|Protocol]]==<br />
<br />
==Initial Idea==<br />
<br />
===<STRIKE>Diet</STRIKE>===<br />
*[[Team:Osaka/Diet-09.07.09|09.07.09 (Japanese only)]]<br />
<br />
===Sound/Illumination===<br />
*[[Team:Osaka/Sound-09.07.09|09.07.09 (Japanese only)]]<br />
<br />
===<STRIKE>Terra forming</STRIKE>===<br />
*[[Team:Osaka/Terra-09.07.09|09.07.09 (Japanese only)]]<br />
<br />
==[[Team:Osaka/Brainstorming|Brainstorming]]==<br />
*[[Team:Osaka/Brainstorming0801|Art 08/01 (Japanese only)]]<br />
<br />
</div></div>TOHRU