Difference between revisions of "Bacterial Transformation : The Process"

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[[File:IMG_5912_-Desktop_Resolution-.JPG|170px|thumb|right|1.The eppendorf tubes being labeled]]
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[[File:DSC_8084.jpg|170px|thumb|right|2.The dry DNA being extracted]]
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File:IMG_5912_-Desktop_Resolution-.JPG|1.The eppendorf tubes being labeled
[[File:DSC_8082.jpg|170px|thumb|none|3.]]
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File:DSC_8084.jpg|2.The dry DNA being extracted
[[File:DSC_8122.jpg|170px|thumb|right|4.]]
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File:DSC_8082.jpg|3.
[[File:DSC_8120.jpg|170px|thumb|none|4.The four eppendorf tubes being cooled]]
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[[File:DSC_8130.jpg|170px|thumb|right|5.]]
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File:DSC_8120.jpg|The four eppendorf tubes being cooled
[[File:DSC_8139.jpg|170px|thumb|none|6.]]
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[[File:DSC_8164.jpg|170px|thumb|right|8.]]
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[[File:IMG_5948_-Desktop_Resolution-.JPG|170px|thumb|none|9.]]
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[[File:DSC_8176.jpg|170px|thumb|right|10.]]
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'''What are we doing?'''
 
'''What are we doing?'''
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'''How are we doing this?'''
 
'''How are we doing this?'''
  
We start by gathering the materials we need:
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We start by gathering the '''materials we need''':
  
 
1. '''Eppendorf tubes'''
 
1. '''Eppendorf tubes'''
 +
 
  The tiny plastic tubes that are used in labs while dealing with solutions that are extremely less in quantity.Unlike regular test tubes, they come with an easily usable lid and a slightly triangular bottm.
 
  The tiny plastic tubes that are used in labs while dealing with solutions that are extremely less in quantity.Unlike regular test tubes, they come with an easily usable lid and a slightly triangular bottm.
  
 
2. '''LB solution'''
 
2. '''LB solution'''
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Luria Bertani solution; named after a scientist is a source of required nutrients and enables the bacteria to grow.
 
Luria Bertani solution; named after a scientist is a source of required nutrients and enables the bacteria to grow.
  
 
3. '''Dry DNA'''(from the DNA Distribution Kit )
 
3. '''Dry DNA'''(from the DNA Distribution Kit )
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The foreign DNA that we are adding to the bacteria.
 
The foreign DNA that we are adding to the bacteria.
  

Revision as of 05:59, 9 June 2009

What are we doing?

We are attempting to alter the genetic composition of a bacteria by adding foreign genetic material to it. For this , the bacteria needs to be conditioned to take foreign material and make it its own. Now it is this process of 'conditioning' that forms the scientific method described below. This, in scientific terms is called 'Transformation'.

How are we doing this?

We start by gathering the materials we need:

1. Eppendorf tubes

The tiny plastic tubes that are used in labs while dealing with solutions that are extremely less in quantity.Unlike regular test tubes, they come with an easily usable lid and a slightly triangular bottm.

2. LB solution

Luria Bertani solution; named after a scientist is a source of required nutrients and enables the bacteria to grow.

3. Dry DNA(from the DNA Distribution Kit )

The foreign DNA that we are adding to the bacteria.

  1. Four eppendorf tubes were taken and marked lysis, p-tet, +ve and -ve. The positive control had the gene that promoted Ampicillin resistance and the negative control did not. If any colonies started forming in the negative control batch, that would mean the entire batch is contaminated.
  2. The dry DNA was extracted by mixing each part with 15 micro ml of water using a micro-pipette.
  3. 2-3 micro ml of this plasmid solution was then added to 100 micro ml of DH 5-alpha E.Coli cells.
  4. The bacteria were incubated in ice for 40 minutes, then given a heat shock for 30 seconds @ 42 degrees Celsius and then put in ice for 2-5 minutes. The heat shock causes tiny pores in the walls of the bacteria to expand, allowing the plasmids to go through.
  5. The Bacteria were then mixed into 330 micro ml LB (Luria Bertani) which is a source of required nutrients and enables the bacteria to grow.
  6. The bacteria were then left to cultivate for approximately an hour @ 37 degrees Celsius in the shaker.
  7. Centrifuged at room temperature for 3 minutes @ 8000 rpm.
  8. Discard 900 micro ml of the supernatant and dissolve pellets in remaining 100 micro ml.
  9. These were then spread on LB Agar plates containing 100 micro gram per ml Ampicillin.
  10. Plates were incubated at 37 degrees Celsius overnight.