Team:Tokyo Metropolitan/Project/Fiber/Protocol

From 2010.igem.org

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==　Grow up a culture of A.xylinus in media==
==　Grow up a culture of A.xylinus in media==
===　Grow up a culture of A.xylinus in media recommend by Open Wet Ware===
===　Grow up a culture of A.xylinus in media recommend by Open Wet Ware===
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<font size="3"><span style="text-decoration:underline">Material</font></span><br>
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　A.xylinus JCM strain 7664<br>
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<font size="3"><span style="text-decoration:underline">Material</font></span>
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　500 ml of liquid Acetobacter media(Open Wet Ware recommended)<br>
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　<ul><li>A.xylinus JCM strain 7664
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　　-Glucose - 1.0 g <br>
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　<li>500 ml of liquid Acetobacter media(Open Wet Ware recommended)
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　　-Peptone - 2.5 g <br>
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　　<ul><li>Glucose - 1.0 g  
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　　-Yeast extract - 2.5 g <br>
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　　<li>Peptone - 2.5 g  
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　　-Na2HPO4 - 1.35 g <br>
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　　<li>Yeast extract - 2.5 g  
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　　-Citric acid - 0.75 g <br>
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　　<li>Na2HPO4 - 1.35 g  
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　　-Distilled water - 500 ml <br>
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　　<li>Citric acid - 0.75 g  
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　　<li>Distilled water - 500 ml </ul></li></ul>
　(If you are making plates, use the same protocol but add 7.5 g of agar.)<br><br>
　(If you are making plates, use the same protocol but add 7.5 g of agar.)<br><br>
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　<li>A.xylinus will grow well at room temperature in aerobic conditions.</ol>
　<li>A.xylinus will grow well at room temperature in aerobic conditions.</ol>
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===　Grow up a culture of A.xylinus in media recommend by JCM===
===　Grow up a culture of A.xylinus in media recommend by JCM===
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　<li>A.xylinus will grow well at room temperature in aerobic conditions.</ol>
　<li>A.xylinus will grow well at room temperature in aerobic conditions.</ol>
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==　Grow up a culture of E.coli==
==　Grow up a culture of E.coli==
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<font size="3"><span style="text-decoration:underline">Material</font></span><br>
<font size="3"><span style="text-decoration:underline">Material</font></span><br>
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<font size="3"><span style="text-decoration:underline">Equipment</font></span><br>
<font size="3"><span style="text-decoration:underline">Equipment</font></span><br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
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==　Direct PCR==
==　Direct PCR==
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<font size="3"><span style="text-decoration:underline">Material</font></span><br>
<font size="3"><span style="text-decoration:underline">Material</font></span><br>
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　E.coli K12 colony<br>
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　10µM/l forward Primer 5µl<br>
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　10µM/l reverse Primer 5µl<br>
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　10×EX taq buffer 10µl<br>
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　2.5mM each dNTP mixture 10µl<br>
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　EX taq polymerase 1µl<br>
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　milli-Q 71µl<br><br>
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<font size="3"><span style="text-decoration:underline">Equipment</font></span><br>
<font size="3"><span style="text-decoration:underline">Equipment</font></span><br>
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　thermal cycler<br>
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　vortex mixer<br>
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　PCR-tubes<br>
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　pipet<br>
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　pipet tip<br><br>
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<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
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<ol><li>Add and mix above materials to 50μl in each PCR tubes on the ice</li>
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<li>Pick up E.coli K12 cells from its colony and add into the PCR tubes</li>
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<li>Setting tips and elongation in the thermal cycler
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<ul><li>initialization :95°C 3min</li>
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<li>denaturation:96°C 1min</li>
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<li>annealing :55°C 5min</li>
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<li>elongation:72°C 1min</li>
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<p>(30cycles from 96°C 1min to 72°C 1min)</p>
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<li>reaction stop :10°C</li></ul></li></ol>
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==　Electrophoreses PCR productions==
==　Electrophoreses PCR productions==
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<font size="3"><span style="text-decoration:underline">Material</font></span><br>
<font size="3"><span style="text-decoration:underline">Material</font></span><br>
<font size="3"><span style="text-decoration:underline">Equipment</font></span><br>
<font size="3"><span style="text-decoration:underline">Equipment</font></span><br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
<font size="3"><span style="text-decoration:underline">Procedure</font></span><br>
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==　DNA purification from agarose gel with QIAGEN==
==　DNA purification from agarose gel with QIAGEN==
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　<font size="3"><span style="text-decoration:underline">Material</font></span><br>
　<font size="3"><span style="text-decoration:underline">Material</font></span><br>
　　QIAGEN(gel extraction kit)<br>
　　QIAGEN(gel extraction kit)<br>
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　12. centrifuge 15000rpm/1min<br>
　12. centrifuge 15000rpm/1min<br>
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Revision as of 08:52, 29 August 2010



E.coli Fiber Project Protocol


Contents

　Grow up a culture of A.xylinus in media

　Grow up a culture of A.xylinus in media recommend by Open Wet Ware

Material 　
  • A.xylinus JCM strain 7664 　
  • 500 ml of liquid Acetobacter media(Open Wet Ware recommended) 　　
    • Glucose - 1.0 g 　　
    • Peptone - 2.5 g 　　
    • Yeast extract - 2.5 g 　　
    • Na2HPO4 - 1.35 g 　　
    • Citric acid - 0.75 g 　　
    • Distilled water - 500 ml
　(If you are making plates, use the same protocol but add 7.5 g of agar.)

Equipment
　autoclave
　incubator
　scale
　bunsen burner
　flask(1l or500ml)
　plate
　spreader
　pipet
　pipet tip

Procedure 　
  1. Prepare media as outlined (add the materials as above) 　
  2. Autoclave to sterilize media(121°C　20minute). 　
  3. Streak/inoculate A.xylinus onto plates or in media. 　
  4. Incubate cells at 26°C for 2-3 days. 　
  5. If using a freeze dried source of A.xylinus, growth may take up to 4 days.

Note 　
  1. The growth of A.xylinus does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. 　
  2. The growth of A.xylinus is accompanied by the formation of a thick cellulose matrix within the media that must be removed before cells can be pelleted for a miniprep procedure. Simply vortex briefly to break up the cellulose into chunks and remove the cellulose chunks from the media with a pipette while carefully avoiding the removal of cells. 　
  3. A.xylinus will grow well at room temperature in aerobic conditions.

　Grow up a culture of A.xylinus in media recommend by JCM

Material
　A.xylinus JCM strain 7664
　1l of liquid Acetobacter media(JCM recommended)
　　-Glucose - 100g
　　-Yeast extract - 10g
　　-CaCO3 30g
　　-Distilled water - 1000 ml
　(If you are making plates, use the same protocol but add 15g of agar.)

Equipment
　autoclave
　incubator
　scale
　bunsen burner
　flask(1l or500ml)
　plate
　spreader
　pipet
　pipet tip

Procedure 　
  1. Prepare media as outlined (add the materials as above) 　
  2. Autoclave to sterilize media(121°C　20minute). 　
  3. Streak/inoculate A.xylinus onto plates or in media. 　
  4. Incubate cells at 26°C for 2-3 days. 　
  5. If using a freeze dried source of A.xylinus, growth may take up to 4 days.

Note 　
  1. The growth of A.xylinus does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. 　
  2. The growth of A.xylinus is accompanied by the formation of a thick cellulose matrix within the media that must be removed before cells can be pelleted for a miniprep procedure. Simply vortex briefly to break up the cellulose into chunks and remove the cellulose chunks from the media with a pipette while carefully avoiding the removal of cells. 　
  3. A.xylinus will grow well at room temperature in aerobic conditions.

　Grow up a culture of E.coli

Material
Equipment
Procedure

　Direct PCR

Material
　E.coli K12 colony
　10µM/l forward Primer 5µl
　10µM/l reverse Primer 5µl
　10×EX taq buffer 10µl
　2.5mM each dNTP mixture 10µl
　EX taq polymerase 1µl
　milli-Q 71µl

Equipment
　thermal cycler
　vortex mixer
　PCR-tubes
　pipet
　pipet tip

Procedure
  1. Add and mix above materials to 50μl in each PCR tubes on the ice
  2. Pick up E.coli K12 cells from its colony and add into the PCR tubes
  3. Setting tips and elongation in the thermal cycler
    • initialization :95°C 3min
    • denaturation:96°C 1min
    • annealing :55°C 5min
    • elongation:72°C 1min

      • (30cycles from 96°C 1min to 72°C 1min)

    • reaction stop :10°C

　Electrophoreses PCR productions

Material
Equipment
Procedure

　DNA purification from agarose gel with QIAGEN

　Material
　　QIAGEN(gel extraction kit)
　　-QG buffer 300µl
　　-PE buffer700µl
　　-EB buffer 50µl
　　-tube for column
　Equipment
　　centrifuge
　　heating plate
　　pipet
　　pipet tip
　Procedure
　1. cut gel of electrophoreses
　2. add pieces of gel to tubes
　3. take 300µl QG buffer into tubes and dissolve at 50°C
　4. add a solution of QG buffer and gel to tubes for column
　5. centrifuge 15000rpm/1min
　6. throw “flow-thru” away and take 700µl PE buffer
　7. centrifuge 15000rpm/1min
　8. throw flow-thru away
　9. centrifuge 15000rpm/1min
　10. change tube for column
　11. add 50µl of EB buffer (aim to center of tube)
　12. centrifuge 15000rpm/1min

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