Transcript Dancing Naked in the Mind Field

Kary Mullis
Born: December 28, 1944
Nobel Prize in Chemistry (1993) for creating
PCR
Quotes:
“Sometimes in the morning, when it's a good surf, I
go out there, and I don't feel like it's a bad world.”
"What if I had not taken LSD ever; would I have
still invented PCR? I don't know. I doubt it. I
seriously doubt it."
Mullis's 1998 autobiography Dancing Naked in the Mind Field, gives his
account of the commercial development of PCR, as well as providing
insights into his opinions and experiences. In the book, Mullis chronicles
his romantic relationships, use of LSD, synthesis and self-testing of novel
psychoactive substances, belief in astrology and an encounter with an
extraterrestrial in the form of a fluorescent raccoon.
Gene Cloning


Preparing a Gene for
Cloning
Green Florescent
Protein (GFP)

In Vivo Gene Cloning

Plasmid Vectors


Antibiotic Resistance
Markers
Gene Expression
Preparing a Gene for Cloning
1)Double stranded DNA of a gene from a eukaryotic organism contains
introns
2)As a normal part of the cell process of gene expression, transcription
creates a primary RNA molecule
3)The introns are removed by restriction enzymes to form a mature
mRNA that codes for the making of a single protein (animation)
4)The mRNA is extracted from the cell and purified
5)Reverse transcriptase is added which synthesises a single stranded
DNA molecule complementary to the mRNA
6)The second DNA strand is made by using the first as a template, and
adding the enzyme DNA polymerase
In Vivo Gene Cloning
1)Gene of interest (GFP) is isolated and prepared by removal of
introns
2)An appropriate plasmid vector is chosen
3)GFP gene and plasmid are each treated with the same restriction
enzymes to make identical cuts
4)The DNA fragments are mixed together and complimentary ends
are attracted by base-pairing.
5)DNA ligase is added to “glue” the GFP gene into the plasmid
6)The recombinant plasmid is introduced into a bacterial culture.
7)About 1 in 10,000 cells will take up the plasmid
http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.htm
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Plasmid Vector
http://blog.addgene.org/plasmids-101-what-is-a-
Antibiotic Resistance
What did the cells take up?

Plasmid only

Plasmid with insert cloned (GFP)

Foreign DNA from the environment

Nothing
How to determine which bacteria
take up GFP gene



Nothing or Foreign DNA only (no plasmid):
Bacteria do not have Amp Resistance so they
will not grow
Plasmid only (no GFP): Bacterial colonies will
not glow
Plasmid with GFP Clone: Bacterial colonies will
glow green under a UV light
GFP Expressed
Ignore the Red Colonies
Plasmid without GFP
GFP Expressed
GFP Expression
Transgenic Organisms
http://www.glofish.com
/