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Ch. 2A: How Do You Begin to Clone a Gene?. Learning goals. Describe the characteristics of plasmids Explain how plasmids are used in cloning a gene Describe the function of restriction enzymes Explain how to use restriction enzymes to create a recombinant plasmid. Key Ideas. - PowerPoint PPT Presentation
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Ch. 2A: How Do You Begin to Clone a Gene?
Learning goals
Describe the characteristics of plasmids
Explain how plasmids are used in cloning a gene
Describe the function of restriction enzymes
Explain how to use restriction enzymes to create a recombinant plasmid
Key IdeasPlasmids - ideal vectors for genetic engineering
– replicate in the bacteria cell– gene promoter– antibiotic resistance as a selectable marker, – can be transferred into bacteria by conjugation.
Restriction enzymes – key to the creation of a recombinant plasmid. – cut DNA at specific sequences – sticky ends allow strands to join
The Plasmid
pARA-R plasmid
Recombinant plasmid of interest
Bruce Wallace
BamH I
Hind III
pARA-R5,302 bp
PBAD-rfp806 bp
pARA-R construct
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
3’
5’
5’
3’
3’
3’
5’
5’5’
5’3’
3’
Bruce WallaceEngineering the Plasmid: ligation of rfp gene into p-ARA
BamH I
Hind III
BamH I
Hind III
Restriction digest of pARA-R
Recombinant plasmid of interest
pARA-R5,302 bp
Biotech Experience
PBAD-rfp806 bp
Restriction analysis of pARA-R
Restriction fragments after digest with Hind III and BamH I
Biotech Experience
806 bp
BamH IHind III
BamH I Hind III
4,496 bp
Learning goals: lab #4
Describe why it is important to verify products created in the genetic engineering process
Predict the relative speed of DNA restriction fragments and plasmids through a gel during gel electrophoresis
Separate and identify DNA restriction fragments and plasmids using gel electrophoresis
Key Ideas
The multistep process that is used to clone a gene results in multiple products– Need to verify that you have the recombinant plasmid
you need.
DNA fragments and plasmids can be separated by gel electrophoresis.
Loading dye helps monitor the progress of the gel electrophoresis procedure.
DNA ladder helps determine the sizes of unknown pieces of DNA
Gel is stained in order to show the location of the DNA fragments and plasmids.
Review questions
1. Why is it important to have sticky ends?
2. What is the purpose of the restriction enzymes?
3. How do you confirm the uptake of the gene into the plasmid?
Clone That Gene activity
1. Cut the plasmid and the human DNA with the appropriate
restriction enzyme 2. Insert the insulin gene into the plasmid DNA 3. Determine which antibiotic you would use to identify bacteria that have taken in the plasmid
Tips
Reagents should be stored in a freezer until you are ready to prepare them for students. Allow to defrost for 15 minutes before using.
The reagents can be aliquoted up to several days before the lab, then store in the freezer/refrigerator
Vortex and spin enzyme mix and 2.5x RB before aliquoting
video
Tips
Calibrate and mark the controller
“Floatie” marked with team number. Each period has a different color.
Bottom of tubes in the water
Low-tech water bath
Multiple pans allow tubes from different classes to stay separated
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