Team:Cambridge/Protocols/Transformation of E.Coli

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==Transformation of E.coli==
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==Transformation of E.Coli==
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A simple method of transforming competent E.coli cells with your DNA of choice.
A simple method of transforming competent E.coli cells with your DNA of choice.
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===Preparation===
 
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Often you will have to do some steps well in advance of carrying out a protocol.
 
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In this case you must take liquid broth to be autoclaved in good time. You should also turn on the water bath and incubator so they are at the required temperature when you need them, but make sure you turn them off after too.
 
===Theory===
===Theory===
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[[#artificial_transformation|Artificial transformation]] is a process whereby E.coli are made competent and take up DNA from their surroundings. The main steps are chilling the cells to 0&ordm;c with CaCl<sub>2</sub> solution, adding the DNA and then heat shocking the bacteria for a short period of time.
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Artificial transformation is a process whereby E.coli are made competent and take up DNA from their surroundings. The main steps are chilling the cells to 0<sup>o</sup>C with CaCl<sub>2</sub> solution, adding the DNA and heat shocking the bacteria for a short period of time, allowing the cells to recover at 37<sup>o</sup>C and then plate them.
One hypothesis for artificial transformation is that the divalent cation provided by the chilled CaCl<sub>2</sub> solution which is used in creating competent cells promotes an interaction between DNA and lipopolysaccharides which are also negatively charge. Lipopolysaccharides occur in higher densities near the parts of the outer membrane of Gram negative bacteria that are in close association with the inner membrane (zones of adhesion).
One hypothesis for artificial transformation is that the divalent cation provided by the chilled CaCl<sub>2</sub> solution which is used in creating competent cells promotes an interaction between DNA and lipopolysaccharides which are also negatively charge. Lipopolysaccharides occur in higher densities near the parts of the outer membrane of Gram negative bacteria that are in close association with the inner membrane (zones of adhesion).
It is believed that prior to incubation DNA may interact with lipopolysaccharides and then cross the 'least barrier path' at the zones of adhesion described above.
It is believed that prior to incubation DNA may interact with lipopolysaccharides and then cross the 'least barrier path' at the zones of adhesion described above.
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===Preparation===
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Ensure that you have enough sterilized liquid broth (or another medium such as SOC) and agar plates containing the correct antibiotic. Before you begin have a waterbath set to 42<sup>o</sup>C and an incubator at 37<sup>o</sup>C.
===Practice===
===Practice===
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Take care not to disturb the competent E.coli, in other words do not vortex them or place them on ice.
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Take care not to disturb the competent E.coli: do not vortex them or pipette them up and down.
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To a clean Eppendorf tube add
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{| border="1px"
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|Reagent
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|Quantity (&mu;l)
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|Competent E.coli cells
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|50
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|DNA
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|10
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|-
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|}
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Incubate the cells with the DNA at 42&ordm;C for 1 minute.
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Add 250&mu;l of liquid broth (which should have already been autoclaved)
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Incubate at 37&ordm;c for 1 hour.
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:*Thaw competent E.coli cells ( 50 &mu;l in an eppendorf tube, grown to an OD<sub>600</sub> of 0.2 - 0.5) on ice. Let them sit on ice for at least 10 minutes.
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:*Add 1 &mu;l of plasmid and mix gently by stirring with the pipette tip. Use more plasmid solution if it is very weak. We have had success with about 50 ng of plasmid DNA.
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:*Incubate the cells with the DNA at 42&ordm;C for 1 minute.
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:*Add 250 &mu;l of sterile liquid broth.
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Plate 10&mu;l on agar containing an antibiotic (the antibiotic for which resistance is included in the DNA being taken up by the cells) and also plate 100&mu;l on another identical plate. This allows for possibly overcrowding as the next step after this protocol usually requires a single colony to be selected.
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At this stage it is important to maintain sterility to avoid contaminating the liquid broth or the plates. Do not allow the pipette tip to touch anything before pipetting the liquid broth. Leaving the liquid broth bottle on the bench overnight should reveal any contamination in the morning.
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Incubate overnight at 37&ordm;C
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:*Incubate at 37&ordm;C for 1 hour.
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:*Plate 10&mu;l on agar containing an antibiotic (the antibiotic for which the plasmid confers resistance) and also plate 100&mu;l on another identical plate. This allows for possible overcrowding so that single colonies can be selected.
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:*Incubate overnight at 37&ordm;C.
Always keep agar plates upside down so that drips of condensation and falling debris does not contaminate them.
Always keep agar plates upside down so that drips of condensation and falling debris does not contaminate them.
===Safety===
===Safety===
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All equipment (including gloves) that may have come into contact with the bacteria must be autoclaved for decontamination purposes.
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All equipment (including gloves) that may have come into contact with the bacteria must be autoclaved.
===References===
===References===
<div id="artificial_transformation"></div>
<div id="artificial_transformation"></div>
<sup>[http://www.ias.ac.in/currsci/dec102002/1376.pdf]</sup> '''Mechanism of artificial transformation of E. coli with plasmid DNA – Clues from the influence of ethanol'''
<sup>[http://www.ias.ac.in/currsci/dec102002/1376.pdf]</sup> '''Mechanism of artificial transformation of E. coli with plasmid DNA – Clues from the influence of ethanol'''
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Latest revision as of 20:24, 21 September 2011

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OVERVIEW
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Contents

Transformation of E.coli

A simple method of transforming competent E.coli cells with your DNA of choice.

Theory

Artificial transformation is a process whereby E.coli are made competent and take up DNA from their surroundings. The main steps are chilling the cells to 0oC with CaCl2 solution, adding the DNA and heat shocking the bacteria for a short period of time, allowing the cells to recover at 37oC and then plate them.

One hypothesis for artificial transformation is that the divalent cation provided by the chilled CaCl2 solution which is used in creating competent cells promotes an interaction between DNA and lipopolysaccharides which are also negatively charge. Lipopolysaccharides occur in higher densities near the parts of the outer membrane of Gram negative bacteria that are in close association with the inner membrane (zones of adhesion).

It is believed that prior to incubation DNA may interact with lipopolysaccharides and then cross the 'least barrier path' at the zones of adhesion described above.

Preparation

Ensure that you have enough sterilized liquid broth (or another medium such as SOC) and agar plates containing the correct antibiotic. Before you begin have a waterbath set to 42oC and an incubator at 37oC.

Practice

Take care not to disturb the competent E.coli: do not vortex them or pipette them up and down.

  • Thaw competent E.coli cells ( 50 μl in an eppendorf tube, grown to an OD600 of 0.2 - 0.5) on ice. Let them sit on ice for at least 10 minutes.
  • Add 1 μl of plasmid and mix gently by stirring with the pipette tip. Use more plasmid solution if it is very weak. We have had success with about 50 ng of plasmid DNA.
  • Incubate the cells with the DNA at 42ºC for 1 minute.
  • Add 250 μl of sterile liquid broth.

At this stage it is important to maintain sterility to avoid contaminating the liquid broth or the plates. Do not allow the pipette tip to touch anything before pipetting the liquid broth. Leaving the liquid broth bottle on the bench overnight should reveal any contamination in the morning.

  • Incubate at 37ºC for 1 hour.
  • Plate 10μl on agar containing an antibiotic (the antibiotic for which the plasmid confers resistance) and also plate 100μl on another identical plate. This allows for possible overcrowding so that single colonies can be selected.
  • Incubate overnight at 37ºC.

Always keep agar plates upside down so that drips of condensation and falling debris does not contaminate them.

Safety

All equipment (including gloves) that may have come into contact with the bacteria must be autoclaved.

References

[http://www.ias.ac.in/currsci/dec102002/1376.pdf] Mechanism of artificial transformation of E. coli with plasmid DNA – Clues from the influence of ethanol Back to Protocols

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