Team:KIT-Kyoto/Notebook/Protocol

From 2011.igem.org

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<table border=0 width="580px" align=left><tr><td>This page lists all the protocols used in our project.</td></tr></table></td></tr></table>
<table border=0 width="580px" align=left><tr><td>This page lists all the protocols used in our project.</td></tr></table></td></tr></table>
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</body></html>
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<b>LB medium</b>
-
 
+
:<table border=1 width="200px">
 +
:<tr><td width="150px" align=center>bacto tryptone</td><td width="50px" align=right>10 g</td></tr>
 +
:<tr><td align=center>bacto yeast evtract</td><td align=right>5 g</td></tr>
 +
:<tr><td align=center>NaCl</td><td align=right>10 g</td></tr>
 +
:</table>
 +
<br>
 +
<br>
 +
<b>LB plate</b>
 +
:<table border=1 width="170px">
 +
:<tr><td width="100px" align=center>LB medium</td><td width="70px" align=right>&nbsp;</td></tr>
 +
:<tr><td align=center>bacto-agar</td><td align=right>15 g/l</td></tr>
 +
:</table>
 +
:↓Add 15g of bacto-agar to 1L of LB medium then sterilize it by autoclave.
 +
:↓If necessary, the antibiotics can be added after the temperature of LB-agar become below 65°C.
 +
:↓Twenty to twenty-five ml of LB-agar should be poured to the disposable sterilized dishes.
 +
<br>
 +
<br>
 +
<b>Transformation</b>
 +
:↓Dissolve the competent cells on ice.
 +
:↓Prepare some iced 1.5ml tubes and pour competent cells for 100µl into the tubes.
 +
:↓Add 1~5µl of DNA solution to the 100µl of competent cells, then incubate for 30 minutes on ice.
 +
:↓Heat shock for 45 seconds at 42°C.
 +
:↓Add 900 µl of soc medium into each tube.
 +
:↓Incubate with shaking at 37°C for 1 hour.
 +
:↓Then spread on the LB plate containing an appropriate antibiotic.
 +
:↓Incubate at 37°C for 16 hours.
 +
<br>
 +
<br>
 +
<b>Alkali SDS</b>
 +
:↓Spread E.coli onto the LB plate (+amp),and incubate it at 37°C(API2-MALT1 for 30°C) for 16 hours.
 +
:↓Pick up the single colony from the plate.
 +
:↓Incubate it into the 2ml LB medium, and shake the tubes at 37°C(API2-MALT1 for 30°C) for hours.
 +
:↓Harvest the bacterial cells by centrifugation at 4°C for 5 minutes (5,000 x g).Discard the supernatant.
 +
:↓Add 100µl of iced SolutionⅠ to the tubes,and mix them using vortex.
 +
:↓Add 200µl of iced SolutionⅡ to the tubes,and mix them sharply not using vortex.
 +
:↓Let them stand on ice for 5 minutes.
 +
:↓Add 150µl of iced Solution Ⅲ to the tubes,and mix them sharply but not using vortex.
 +
:↓Let them stand on ice for 5 minutes.
 +
:↓Centrifuge them at 4°C for 10 minutes (12,000 x g).
 +
:↓Transfer supernatant into the tubes.
 +
:↓Add 450µl of Isopropanol and mix them well.Let them for 15 minutes.Then recover the DNA by centrifugation at 12,000 x g for 30 minutes at 25°C.
 +
<br>
 +
<br>
 +
<b>Ligation</b>
 +
:Refer to following table, prepare a reaction solution.
 +
:<table border=1 width="200px">
 +
:<tr><td width="130px" align=center>insert</td><td width="70px" align=right>0.5 µl</td></tr>
 +
:<tr><td align=center>vector</td><td align=right>0.5 µl</td></tr>
 +
:<tr><td align=center>2 x Buffer</td><td align=right>2.5 µl</td></tr>
 +
:<tr><td align=center>T4 ligase</td><td align=right>0.5 µl</td></tr>
 +
:<tr><td align=center>H<sub>2</sub>O</td><td align=right>1.0 µl</td></tr>
 +
:<tr><td>&nbsp;</td><td align=right>total 5 µl</td></tr>
 +
:</table>
 +
:Incubate it for 30min at 16 ℃.
 +
<br>
 +
<br>
 +
<b>PCR</b>
 +
:Add all reagents in a PCR tube.<br>
 +
:Based on primers, set an appropriate annealing temperature.
 +
<br>
 +
<br>
 +
<b>アガロース電気泳動</b>
 +
:↓Prepare a 1% (w/v) agarose gel.
 +
:↓Add 10 µl of 6x loading dye to each 50 µl of digested solutions.
 +
:↓Load each of 60 µL samples and DNA maker in wells.
 +
:↓Run at 50 V and 60min.
 +
:↓Stain in EtBr solution for 10min.
 +
<br>
 +
<br>
 +
<b>コンピ</b>
 +
:↓Streak E.coli on LB plate and incubate for 16h.
 +
:↓Pick a single colony up, transfer it into 250 ml of SOB medium in a flask. Incubate it at 18°C with shaking.
 +
:↓When it reaches an OD600 of 0.4 to 0.8, transfer it on an ice for 10 min.
 +
:↓Harvest cells by centrifugation(4.5 x 10<sup>3</sup> g) for 10min at 4°C.
 +
:↓Remove the supernatant and resuspend the cells in 84 ml of ice-cold TB.
 +
:↓Keep it on ice for 10min.
 +
:↓Harvest cells by centrifugation(5 x 10<sup>3</sup> g) for 10min at 4°C.
 +
:↓Remove the supernatant and resuspend the cells in 40 ml of ice-cold TB.
 +
:↓Add 1.5 ml of DMSO(7%) and keep it on ice for 10min.
 +
:↓Pour each of 220 µl into chilled eppendorf tubes. Make sure to close tightly and then flash freeze in liquid nitrogen and store at -80°C.
 +
<br>
 +
<br>
</div>
</div>

Revision as of 14:52, 5 October 2011



Home Team Project Parts Notebook Safety Human Practice Attributions


Home > Notebook > Protocol

This page lists all the protocols used in our project.

LB medium

bacto tryptone10 g
bacto yeast evtract5 g
NaCl10 g



LB plate

LB medium 
bacto-agar15 g/l
↓Add 15g of bacto-agar to 1L of LB medium then sterilize it by autoclave.
↓If necessary, the antibiotics can be added after the temperature of LB-agar become below 65°C.
↓Twenty to twenty-five ml of LB-agar should be poured to the disposable sterilized dishes.



Transformation

↓Dissolve the competent cells on ice.
↓Prepare some iced 1.5ml tubes and pour competent cells for 100µl into the tubes.
↓Add 1~5µl of DNA solution to the 100µl of competent cells, then incubate for 30 minutes on ice.
↓Heat shock for 45 seconds at 42°C.
↓Add 900 µl of soc medium into each tube.
↓Incubate with shaking at 37°C for 1 hour.
↓Then spread on the LB plate containing an appropriate antibiotic.
↓Incubate at 37°C for 16 hours.



Alkali SDS

↓Spread E.coli onto the LB plate (+amp),and incubate it at 37°C(API2-MALT1 for 30°C) for 16 hours.
↓Pick up the single colony from the plate.
↓Incubate it into the 2ml LB medium, and shake the tubes at 37°C(API2-MALT1 for 30°C) for hours.
↓Harvest the bacterial cells by centrifugation at 4°C for 5 minutes (5,000 x g).Discard the supernatant.
↓Add 100µl of iced SolutionⅠ to the tubes,and mix them using vortex.
↓Add 200µl of iced SolutionⅡ to the tubes,and mix them sharply not using vortex.
↓Let them stand on ice for 5 minutes.
↓Add 150µl of iced Solution Ⅲ to the tubes,and mix them sharply but not using vortex.
↓Let them stand on ice for 5 minutes.
↓Centrifuge them at 4°C for 10 minutes (12,000 x g).
↓Transfer supernatant into the tubes.
↓Add 450µl of Isopropanol and mix them well.Let them for 15 minutes.Then recover the DNA by centrifugation at 12,000 x g for 30 minutes at 25°C.



Ligation

Refer to following table, prepare a reaction solution.
insert0.5 µl
vector0.5 µl
2 x Buffer2.5 µl
T4 ligase0.5 µl
H2O1.0 µl
 total 5 µl
Incubate it for 30min at 16 ℃.



PCR

Add all reagents in a PCR tube.
Based on primers, set an appropriate annealing temperature.



アガロース電気泳動

↓Prepare a 1% (w/v) agarose gel.
↓Add 10 µl of 6x loading dye to each 50 µl of digested solutions.
↓Load each of 60 µL samples and DNA maker in wells.
↓Run at 50 V and 60min.
↓Stain in EtBr solution for 10min.



コンピ

↓Streak E.coli on LB plate and incubate for 16h.
↓Pick a single colony up, transfer it into 250 ml of SOB medium in a flask. Incubate it at 18°C with shaking.
↓When it reaches an OD600 of 0.4 to 0.8, transfer it on an ice for 10 min.
↓Harvest cells by centrifugation(4.5 x 103 g) for 10min at 4°C.
↓Remove the supernatant and resuspend the cells in 84 ml of ice-cold TB.
↓Keep it on ice for 10min.
↓Harvest cells by centrifugation(5 x 103 g) for 10min at 4°C.
↓Remove the supernatant and resuspend the cells in 40 ml of ice-cold TB.
↓Add 1.5 ml of DMSO(7%) and keep it on ice for 10min.
↓Pour each of 220 µl into chilled eppendorf tubes. Make sure to close tightly and then flash freeze in liquid nitrogen and store at -80°C.