Baby cell column

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The baby cell column is a way of getting synchronized bacterial cells (E. coli) which are at more or less the same point in their cell cycel.
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{{back to protocols}}
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The baby cell column is a way of getting synchronized bacterial cells (''E. coli'') which are at more or less the same point in their cell cycel.
==Basic idea==
==Basic idea==
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==Methode==
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===Preperation of cells===
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* grow strain NK9386 in 5 ml of your medium (the same as used for the bady cell column) over night
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* start a 1 l culture in 2 l plastic flasks (the sticky cells would stick to a glas flask!)
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* at OD ~0.2 add 1 ml of 100mM IPTG and let grow for one more hour at low shaking (100rpm)
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* centrifuge 10 min at 3000rpm at room temperature
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* leave about 25ml medium on the pellet and resuspend cells
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* add cells to the glass beads on the column (see below)
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===Preparation of column===
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* put 10g of glass beads on the column and set up tubing, pump etc.
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* let system run with about 5ml/min flow rate and 70% ethanol for 10 min
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* let system run with dest. water for 10 min
 +
* let system run with medium for 5 min
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* stop pump and close tube comming behind the column
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* add cells to column and let stand for 15 min
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* let slowly run not attached cells of the column
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* add 50 ml fresh medium (by hand) to the column and let run thru quickly to wash
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* add Whatman filter paper on top of the glass beads
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* start medium folw with 5ml/min
==References==
==References==
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#Bates2005MolMicro pmid=15978072
#Bates2005MolMicro pmid=15978072
</biblio>
</biblio>
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[[Category:Protocol]]  [[Category:Needs attention]]

Current revision

back to protocols

The baby cell column is a way of getting synchronized bacterial cells (E. coli) which are at more or less the same point in their cell cycel.

Contents

Basic idea

Bacteria are attached to glass beads with there flagella. The protein coding for a specailly sticy flagella is introduced on the chromosome behind an inducible promotor. The glass beads with the cells are than put on a column and medium without inductor is run thru it. The new born cells will than not have a flagella and washed of with the medium. Such cells will be "babies" and at the beginning of the cell cycel.

Material

For the set up of the column one needs the following material

Part Company and cat. numberPrice Notes Link
1x Rotilabo®-Luer-tubing connectors, Luer-fitting: LLM, for 2,4mm tubing, 10 pices Roth CT59.1 9.95 € http://www.carlroth.com
1x Rotilabo®-Luer-tubing connectors, Luer-fitting: LLF, for 2,4mm tubing, 10 pices Roth CT63.1 9.95 €
1x Rotilabo®-Mini hose reducers, straight , 2,4 – 4,8 mm, 10 pices Roth CT46.111.95 €
1x Rotilabo®-stoppers with hole, 23-29- 5mm hole, 10 pices Roth Y852.118,90 €
3x Rotilabo®-stand rods 600mm Roth 2378.18,95 €
2x Rotilabo®-retort stand basesRoth 2369.115.20 €
7x Rotilabo®-cross double boss headRoth 2194.17.95 €
5x Rotilabo®-stand clamps, made of malleable iron, 40mmRoth 1995.19.45 €
1x Rotilabo®-tube clamps, Polysulfone (autoclavable)Roth T764.1 32.80 €
1x Corning® Erlenmeyer flask polycarbonate baffled flasks capacity 2000 mL, polypropylene cap (vented), package with 6 picesSigma CLS431256-6EA300 €http://www.sigmaaldrich.com

Methode

Preperation of cells

  • grow strain NK9386 in 5 ml of your medium (the same as used for the bady cell column) over night
  • start a 1 l culture in 2 l plastic flasks (the sticky cells would stick to a glas flask!)
  • at OD ~0.2 add 1 ml of 100mM IPTG and let grow for one more hour at low shaking (100rpm)
  • centrifuge 10 min at 3000rpm at room temperature
  • leave about 25ml medium on the pellet and resuspend cells
  • add cells to the glass beads on the column (see below)

Preparation of column

  • put 10g of glass beads on the column and set up tubing, pump etc.
  • let system run with about 5ml/min flow rate and 70% ethanol for 10 min
  • let system run with dest. water for 10 min
  • let system run with medium for 5 min
  • stop pump and close tube comming behind the column
  • add cells to column and let stand for 15 min
  • let slowly run not attached cells of the column
  • add 50 ml fresh medium (by hand) to the column and let run thru quickly to wash
  • add Whatman filter paper on top of the glass beads
  • start medium folw with 5ml/min

References

  1. Bates D, Epstein J, Boye E, Fahrner K, Berg H, and Kleckner N. . pmid:15978072. PubMed HubMed [Bates2005MolMicro]
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