cloning dna into plasmid vectors and sequencing
Download
Skip this Video
Download Presentation
Cloning DNA into Plasmid Vectors and Sequencing.

Loading in 2 Seconds...

play fullscreen
1 / 14

Cloning DNA into Plasmid Vectors and Sequencing. - PowerPoint PPT Presentation


  • 211 Views
  • Uploaded on

Cloning DNA into Plasmid Vectors and Sequencing. ABE Workshop 2007 Josh Nelson 26 – Jun – 2007. What is Cloning?. Making multiple identical copies of a gene This can be placed into a vector This in turn can be placed into a host bacteria

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Cloning DNA into Plasmid Vectors and Sequencing.' - merv


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
cloning dna into plasmid vectors and sequencing

Cloning DNA into Plasmid Vectors and Sequencing.

ABE Workshop 2007

Josh Nelson

26 – Jun – 2007

what is cloning
What is Cloning?
  • Making multiple identical copies of a gene
  • This can be placed into a vector
  • This in turn can be placed into a host bacteria
  • Modern plasmid vectors are designed to make the process fast and easy
  • Inclusions of antibiotics and other screening methods ensure correct gene cloning
steps in the cloning process
Steps in the Cloning Process

Choose a vector

  • Isolate Protein
isolation of protein
Isolation of Protein
  • Amino acid sequencing
  • Purify protein
  • Sequence protein
  • Design primer
  • Run PCR
  • Result: DNA of interest

PDI

Isolate Protein

  • Start with PDI
  • Obtain sequence from database
  • Make primers
  • Run PCR
steps in the cloning process1
Steps in the Cloning Process
  • Isolate Protein
  • Choose and Prepare Vector
choosing and preparing the vector
Choosing and Preparing the Vector
  • pCR 4 – TOPO
    • Small plasmid
    • LacZ – ccdB
      • Selective for ccdB gene
  • Insert PCR Product
    • A overhang
    • Vector has T overhang
  • Ligation
    • Topoisomerase
steps in the cloning process2
Steps in the Cloning Process
  • Isolate Protein
  • Choose and Prepare Vector
  • Transform intoHost
transforming into expression host
Transforming into Expression Host
  • E. Coli Cells
    • Soak in CaCl2
      • Make them competent
  • Insert vector
    • Heat shock
  • Spread on culture plate
    • Kanamycin added for selection
  • Incubate
steps in the cloning process3
Steps in the Cloning Process
  • Isolate Protein
  • Choose and Prepare Vector
  • Transform into Host
  • Extraction of Cloned Gene
extraction of cloned gene
Extraction of Cloned Gene

White

Blue/white?

  • Choose colonies
    • White
  • Place in LB medium
    • Contains Ampicillin & Kanamycin
    • Incubate
  • Isolate cloned gene
    • Cell pellet
    • Gene pellet
  • Run gel to confirm you cloned correct gene

Blue

steps in cloning the process
Steps in Cloning the Process
  • Isolate Protein
  • Choose and Prepare Vector
  • Transform into Host
  • Extraction of Cloned Gene
  • Sequence
sequencing
Sequencing
  • Add primers to DNA
    • T7 upstream
    • T3 downstream
  • Dideoxynucleotides
    • Terminates synthesis
  • Fluorescent Dyes
    • Help in fast sequencing
    • Dyes different weights
  • Polyacrylamide Gel Electrophoresis
results of sequencing
Results of Sequencing
  • One was PDI 1
    • White colony
  • One was vector
    • Base pairs between 1550 and 2300
    • ccdB gene not expressed
my application
My Application
  • Shrimp aquaculture
  • Microbial community
  • Build up of ammonia
  • Clone gene which expresses desired trait
ad