hey kim, this site shows the kids everything! ny ‘09 use this for the prelab stuff!! ...
TRANSCRIPT
• Hey Kim, this site shows the kids everything! NY ‘09 use this for the prelab stuff!!
• http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/Bio%20100/Bio%20100%20Laboratory/Bacterial%20Transformation/transformation.htm
AP Bio
Lab #6B
pGLO Bacterial Transformation
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Aequorea victoria
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has a GFP gene= a gene that codes for Green Fluorescent Protein
has been added to the pGLO plasmid
GFP = a gene that codes for Green Fluorescent Protein
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Lets look @ the Plasmid map you will be inserting:
ori = origin of replication
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araC = operon promoter site remember: this is where RNA polymerase binds to the DNA
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Plasm
id m
ap:
bla = gene that codes for B-lactamase… a protein that makes resistance to ampicillin (it breaks the
ampicillin down!!)
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MATERIALS
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LB/amp LB/amp
LB/amp/ara LB
+
-
4 plates +1 STARTER PLATE
• +pGLO LB/amp w/ plasmid
• +pGLO LB/amp/ara w/ plasmid
• -pGLO LB/amp w/o plasmid
• -pGLO LB w/o plasmid
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LB/amp LB/amp
LB/amp/ara LB
+-
1 STARTER PLATE
• E. coli on Luria
Broth (LB) media
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Know your pipettor!!
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HERE WE GO…• In order to induce transformation competence, bacterial cells are first treated with an ice-cold solution of calcium chloride.
• It is believed that the positive calcium chloride binds to the negatively charged DNA strands.
• It then binds to the cell membrane
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Transferring Bacteria
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• Organisms are transferred by using a sterile loop and reaching in from the side while keeping the plate covered as much as possible. This technique minimizes the risk of contamination from above.
• Immerse a sterile loop into the bottle containing plasmid DNA. When the center of the loop is coated with a soap-like film, transfer it to the “+” DNA microtube.
• Use a new sterile loop to transfer a second loopful of plasmid DNA into the same (+ DNA) microtube.
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• The - DNA microtube will not receive any plasmid DNA.
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4 plates
• +pGLO LB/amp + = plasmid added
• +pGLO LB/amp/ara
+ = plasmid added• -pGLO LB/amp
- = no plasmid
• -pGLO LB
- = no plasmid
The Arabinose Operon
araCaraB araB araB
These code for digestive enzymes that break down arabinose sugar
arabinose sugar
araCaraB araB araB
RNA polymerase
araCaraB araB araB
RNA polymerase
mRNA
The Expression of GFP
araCGFP
Our plasmid’s ara Operon has been genetically altered, the regulatory
genes have been replaced w/ the GFP gene from the jelleyfish
arabinose sugar
araC RNA polymerase
araCaraB
RNA polymerase
mRNA
GFP
Results
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