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Team:Tokyo Metropolitan/Project/Fiber/Protocol
From 2010.igem.org
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scale<br> | scale<br> | ||
bunsen burner<br> | bunsen burner<br> | ||
| - | + | flask(1l or500ml)<br> | |
plate<br> | plate<br> | ||
spreader<br> | spreader<br> | ||
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</html> | </html> | ||
=== Grow up a culture of A.xylinus in media recommend by JCM=== | === Grow up a culture of A.xylinus in media recommend by JCM=== | ||
| - | <html> | + | <html><body width="750"> |
| - | <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> | + | A.xylinus JCM strain 7664<br> |
| - | <font size="3"><span style="text-decoration:underline">Procedure</font></span><br> | + | 1l of liquid Acetobacter media(JCM recommended)<br> |
| + | -Glucose - 100g <br> | ||
| + | -Yeast extract - 10g <br> | ||
| + | -CaCO3 30g <br> | ||
| + | -Distilled water - 1000 ml <br> | ||
| + | (If you are making plates, use the same protocol but add 15g of agar.)<br><br> | ||
| + | |||
| + | <font size="3"><span style="text-decoration:underline">Equipment</font></span><br> | ||
| + | autoclave<br> | ||
| + | incubator<br> | ||
| + | scale<br> | ||
| + | bunsen burner<br> | ||
| + | flask(1l or500ml)<br> | ||
| + | plate<br> | ||
| + | spreader<br> | ||
| + | pipet<br> | ||
| + | pipet tip<br><br> | ||
| + | <font size="3"><span style="text-decoration:underline">Procedure</font></span> | ||
| + | <ol><li>Prepare media as outlined (add the materials as above) | ||
| + | <li>Autoclave to sterilize media(121°C 20minute). | ||
| + | <li>Streak/inoculate A.xylinus onto plates or in media. | ||
| + | <li>Incubate cells at 26°C for 2-3 days. | ||
| + | <li>If using a freeze dried source of A.xylinus, growth may take up to 4 days.</ol><br> | ||
| + | <font size="3"><span style="text-decoration:underline">Note</font></span> | ||
| + | <ol><li>The growth of A.xylinus does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance. | ||
| + | <li>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. | ||
| + | <li>A.xylinus will grow well at room temperature in aerobic conditions.</ol> | ||
| + | </body> | ||
</html> | </html> | ||
== Grow up a culture of E.coli== | == Grow up a culture of E.coli== | ||
Revision as of 19:22, 28 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
MaterialA.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
- Prepare media as outlined (add the materials as above)
- Autoclave to sterilize media(121°C 20minute).
- Streak/inoculate A.xylinus onto plates or in media.
- Incubate cells at 26°C for 2-3 days.
- If using a freeze dried source of A.xylinus, growth may take up to 4 days.
Note
- The growth of A.xylinus does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance.
- 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.
- A.xylinus will grow well at room temperature in aerobic conditions.
Grow up a culture of A.xylinus in media recommend by JCM
MaterialA.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
- Prepare media as outlined (add the materials as above)
- Autoclave to sterilize media(121°C 20minute).
- Streak/inoculate A.xylinus onto plates or in media.
- Incubate cells at 26°C for 2-3 days.
- If using a freeze dried source of A.xylinus, growth may take up to 4 days.
Note
- The growth of A.xylinus does not give a cloudy appearance in the media, the media will remain transparent to slightly translucent in appearance.
- 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.
- A.xylinus will grow well at room temperature in aerobic conditions.
Grow up a culture of E.coli
Material
Equipment
Procedure
Direct PCR
Material
Equipment
Procedure
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
