Transcript Lab #6B Transformation

• 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%20Tran
sformation/transformation.htm
AP Bio
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Lab #6B
pGLO Bacterial
Transformation
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
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GFP = a gene that codes for
Green Fluorescent Protein
Lets look @ the Plasmid map you
will be inserting:
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ori = origin of replication
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araC = operon promoter site
remember: this is where RNA
polymerase binds to the DNA
Plasmid map:
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bla = gene that codes for
B-lactamase… a protein that makes
resistance to ampicillin (it breaks the
ampicillin down!!)
MATERIALS
LB/amp LB/amp
LB/amp/ara
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+
LB
4 plates +
1 STARTER PLATE
• +pGLO LB/amp
w/ plasmid
LB/amp LB/amp
LB/amp/ara
LB
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+
• +pGLO LB/amp/ara
w/ plasmid
• -pGLO LB/amp
w/o plasmid
• -pGLO LB
w/o plasmid
1 STARTER PLATE
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• E. coli
on
Luria
Broth
(LB)
media
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 soaplike 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.
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
These code for digestive enzymes
that break down arabinose sugar
araC
araB
araB
araB
arabinose sugar
araC
RNA
polymerase
araB
araB
araB
araB
RNA
araB
polymerase
araB
mRNA
araC
The Expression of GFP
Our plasmid’s ara Operon has been
genetically altered, the regulatory
genes have been replaced w/ the
GFP gene from the jelleyfish
araC
GFP
arabinose sugar
araC
RNA
polymerase
GFP
mRNA
araC
RNA
araB
polymerase
Results
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