Hey Kim, this site shows the kids everything! NY ‘09 use this for the prelab stuff!! ...
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![Page 1: Hey Kim, this site shows the kids everything! NY ‘09 use this for the prelab stuff!! Michael.Gregory/files/Bio%20100/Bio%](https://reader036.fdocuments.us/reader036/viewer/2022062408/56649f325503460f94c4e157/html5/thumbnails/1.jpg)
• 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
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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
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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
+
-
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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
+-
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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.
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• 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
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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
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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
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Results
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