From 2010.igem.org
E.coli Fiber Project Protocol
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
- 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
Material
- A.xylinus JCM strain 7664
- 500 ml of liquid Acetobacter media(Open Wet Ware recommended)
- Glucose - 100g
- Yeast extract - 10g
- CaCO3 30g
- Distilled water - 1000 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 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
- Add and mix above materials to 50μl in each PCR tubes on the ice
- Pick up E.coli K12 cells from its colony and add into the PCR tubes
- Setting tips and elongation in the thermal cycler
|
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
|