Team:Cambridge/Protocols/Overnight Culture

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==E. Coli Cell Culture==
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Method used to prepare a culture of E. coli in liquid medium for DNA extraction.
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==E. coli Cell Culture==
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===Theory===
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Method to prepare a culture of E. coli in liquid medium for DNA extraction.
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===Method===
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The bacteria are cultured in liquid medium in order to allow them to grow freely, amplifying the DNA as much as possible, and the DNA is extracted while the bacteria are still in their mid-exponential growth phase to maximise the proportion of living cells (with intact DNA). Antibiotics are added to the growth medium to ensure that only the strain of bacteria with the required DNA can grow, since these will have been designed to have specific antibiotic resistance. Centrifuging the bacteria lyses the cells, releasing the DNA so that they are exposed to the reagents used in the [https://2011.igem.org/Team:Cambridge/Protocols/Mini_Prep miniprep] protocol.
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This is the method for overnight preparation of a 30ml cell culture of ampicillin-resistant E. coli. For a smaller culture volumes must be adjusted and as well as the incubation time. Most transgenic bacteria should have resistance to some antibiotic which should be substituted for ampicillin.
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===Practice===
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This is the method for overnight preparation of a 30ml cell culture of ampicillin-resistant E. coli. For a smaller culture volumes must be adjusted and as well as the incubation time. Most transgenic bacteria should have resistance to some antibiotic, which should be substituted for ampicillin in this protocol.
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;  '''1.'''
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*Media preparation: Add 1 μl of 1000x concentrated ampicillin for each 1 ml of liquid broth. Be careful to keep the medium sterile especially if not adding an antibiotic, to avoid contamination.
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:Media preparation: Add 1 μl of 1000x concentrated ampicillin for each 1 ml of liquid broth. Be careful to keep the medium sterile especially if not adding an antibiotic, to avoid contamination.
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;  '''2.'''
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*Label on the plates the colonies you wish to culture.
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:Label on the plates the colonies you wish to culture.
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;  '''3.'''
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*Pour 30 ml of the prepared medium into a falcon tube labelled to match the desired colony.
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:Pour 30 ml of the prepared medium into a falcon tube labelled to match the desired colony.
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;  '''4.'''
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*Using a sterile loop, transfer the colony to the liquid medium in the tube.
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:Using a sterile loop, transfer the colony to the liquid medium in the tube.
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;  '''5.'''
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*Incubate for 14-16h in a shaking incubator at 37 degrees.
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:Incubate for 14-16h in a shaking incubator at 37 degrees.
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;  '''6.'''
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*To pellet the bacteria, centrifuge for at least 15 minutes at 3000 rpm, making sure that the tubes are balanced.
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:To pellet the bacteria, make sure the tubes are balanced and centrifuge for at least 15 minutes at 3000 rpm.
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===Safety===
===Safety===
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All materials coming into contact with transgenic E. coli must be autoclaved.
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All materials that come into contact with transgenic E. coli must be autoclaved.
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Latest revision as of 20:25, 21 September 2011

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Contents

E. Coli Cell Culture

Method used to prepare a culture of E. coli in liquid medium for DNA extraction.

Theory

The bacteria are cultured in liquid medium in order to allow them to grow freely, amplifying the DNA as much as possible, and the DNA is extracted while the bacteria are still in their mid-exponential growth phase to maximise the proportion of living cells (with intact DNA). Antibiotics are added to the growth medium to ensure that only the strain of bacteria with the required DNA can grow, since these will have been designed to have specific antibiotic resistance. Centrifuging the bacteria lyses the cells, releasing the DNA so that they are exposed to the reagents used in the miniprep protocol.

Practice

This is the method for overnight preparation of a 30ml cell culture of ampicillin-resistant E. coli. For a smaller culture volumes must be adjusted and as well as the incubation time. Most transgenic bacteria should have resistance to some antibiotic, which should be substituted for ampicillin in this protocol.

  • Media preparation: Add 1 μl of 1000x concentrated ampicillin for each 1 ml of liquid broth. Be careful to keep the medium sterile especially if not adding an antibiotic, to avoid contamination.
  • Label on the plates the colonies you wish to culture.
  • Pour 30 ml of the prepared medium into a falcon tube labelled to match the desired colony.
  • Using a sterile loop, transfer the colony to the liquid medium in the tube.
  • Incubate for 14-16h in a shaking incubator at 37 degrees.
  • To pellet the bacteria, centrifuge for at least 15 minutes at 3000 rpm, making sure that the tubes are balanced.

Safety

All materials that come into contact with transgenic E. coli must be autoclaved.

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