Difference between revisions of "Week Four"

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===June 8th===
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===June 8th- 17th===
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'''''[[A Brief Description of What We Will Do]]'''''
 
  
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[[Preparing the pBAD and Lysis plasmids for gel electrophoresis|The Plasmid preparation process]] | [[The Plasmid Preparation Miniprep Gallery]]
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'''What are we trying to do?'''
  
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We want the bacteria to lyse, to kill itself from the inside.
  
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''' A Brief of what we did: ''' <br/>
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Well then, to do that we had to get a lysis gene in it. The thing is, with the lysis gene needs to be turned on by this specific substance called a '''promoter'''
  
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The plasmids that contained the pBAD and Lysis genes that were prepared were extracted from the bacteria cultures. The unwanted protein around the DNA was then removed. After that the DNA was cleansed of any remaining salt. The plasmids were then cut at certain points such that only the required gene could be extracted. These cut parts were prepared for gel electrophoresis.
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There are two kinds of promoters, ones that make the gene they're attached to , be 'on'  all the time.  
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Then, you have ones that turn on when you want them to, in our case, when you add a mystery enzyme.
  
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'''Our Step-wise Process:'''
  
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===June 9th===
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How we tried to do this:
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[[The June 9th Image Gallery]]
 
  
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Images of the various results attained:
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'''[[Step 1 - Taking dry DNA from wells]]'''
  
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<gallery>
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'''[[Step 2 - Transforming competent cells]]'''
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File:Gelimage090609.png
 
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File:Not working.jpg
 
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File:PBAD-insert f.png
 
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</gallery>
 
  
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'''For June 14th:'''
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'''Step 3 - Picking a single colony.'''
  
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*(Lysis= K112808)
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'''Step 4 - Inoculating broth with Ampicillin-R''' (an antibiotic) and letting it grow for 18 hours.
  
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pBAD- promoter
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'''Step 5 - Using the resulting culture for [[mini-prep.]]''' - ''a process used to purify plasmids and yields clean, usable DNA.''
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Lysis-big construct- gene for cell lysis
 
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Gel image of released pBAD(I0500), after digestion with EcoRI+PstI:
 
  
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Each pBAd and Lysis has 4 restriction enzymes E, X,S, P.
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'''Step 6 - [[Digesting the DNA]]'''
  
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pBAD is digested with EcoRI +SPeI
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'''Step 7 - [[Gel Electrophoresis]]'''
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Lysis is digested with EcoRI + XbaI
 
  
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A small interlink sequence between E and X will break open and spread as one band
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'''Step 8-  [[Ligation]]'''
  
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'''For June 15th:'''
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'''Step 9-  [[Transformation and Inoculation]]'''
  
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These bands will be kept overnight in 37 degrees Celsius.
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===June 9th===
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[[The June 9th Image Gallery]]
  
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Then these will be run through a preparative gel which takes about 3 hours.
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Images of the various results attained:
  
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The next step is to [[elute  the released products]] which wold take 1.5 hours.
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<gallery>
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File:Gelimage090609.png|Agarose gel electrophoresis image of digested K112808
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CIP treatment will be done
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File:Not working.jpg|Failure 1
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[Calf Intestine Phosphate]
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File:PBAD-insert f.png| The pBAD Insert
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This treatment is given to only the vector of Lysis (Process will take 1.5 hour)
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File:Failed Ligation.png| The Failed Ligation Attempt
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This is done so that the vector doesn't self ligate.
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</gallery>
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This enzyme will then be inactivated at 65 degree C.
 
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This vector needs to be purified using column or chloroform
 

Latest revision as of 06:56, 26 June 2009

June 8th- 17th

What are we trying to do?

We want the bacteria to lyse, to kill itself from the inside.

Well then, to do that we had to get a lysis gene in it. The thing is, with the lysis gene needs to be turned on by this specific substance called a promoter

There are two kinds of promoters, ones that make the gene they're attached to , be 'on' all the time. Then, you have ones that turn on when you want them to, in our case, when you add a mystery enzyme.

Our Step-wise Process:

How we tried to do this:

Step 1 - Taking dry DNA from wells

Step 2 - Transforming competent cells

Step 3 - Picking a single colony.

Step 4 - Inoculating broth with Ampicillin-R (an antibiotic) and letting it grow for 18 hours.

Step 5 - Using the resulting culture for mini-prep. - a process used to purify plasmids and yields clean, usable DNA.

Step 6 - Digesting the DNA

Step 7 - Gel Electrophoresis

Step 8- Ligation

Step 9- Transformation and Inoculation

June 9th

The June 9th Image Gallery

Images of the various results attained:

  • Agarose gel electrophoresis image of digested K112808

  • Failure 1

  • The pBAD Insert

  • The Failed Ligation Attempt

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