Transcript Pre-lab 1 and Lab 1 2010 - Sonoma Valley High School

Program Introduction
Chapter 1 – Some Tools of the Trade
Key ideas: Introduction
Genetic engineering allows humans to insert
human DNA into other organisms and then have
these genetically modified organisms make
human proteins. These proteins can be used to
treat a wide variety of diseases and help millions
of people.
The sequence of labs in the Amgen Biotech
Experience mimics the research and
development process used for the recombinant
products that are currently available to treat a
wide range of diseases.
Key ideas
The core of the genetic engineering - carefully planned
changes to DNA lead to production of specific proteins
Genetic disease can be treated using proteins produced
by bacteria whose DNA has been changed by the
addition of the corresponding human gene.
Those who carry out genetic engineering use very
specific tools and have well-honed laboratory skills.
Micropipettes are used to measure and transfer very
small volumes of liquids.
The Steps we will take
1.
Learn to use the equipment
1.
micropipeting & gel electrophoresis
Prepare a plasmid to insert into
bacterium
3. Verify the uptake of gene
4. Transforming bacteria with
recombinant plasmid
2.
Using equipment
 Practice setting volumes on the pipettes
 Aloquoting with pipettes
 Loading, running & reading gels
Adding gene fragments to plasmid
 Use enzymes to cut and splice gene fragments
Verifying gene uptake
 Use gel electrophoresis to verify plasmid has gene
fragment
Transforming E. Coli with a recombinant plasmid
 Transformation & verification on growth plates
Skills & labs for this unit
Using the Micropipette
Video 1 Video 2
Types of Micropipettes
Lock
Pipetting Technique
Hold the
micropipette and
microfuge tubes
at eye level when
loading or
dispensing
samples
Tips
If you are pipetting, you should be holding
the tube.
Common
mistake!
With both elbows
on the table, use
your other hand to
stabilize the
bottom of the
pipette.
Critical Micropipetting Rules
NEVER…use without a tip in place
NEVER…lay it down with sample in the tip
NEVER…let the “plunger” button snap back
Proceed to Student Guide and
Complete Lab 1.1
Loading Gels
Insert pipette tip:
•Under buffer level
•Above gel well
K. Schramm
Different pipetting techniques – stability is the key
Common
Loading errors
Tip should be
above, not in the
well.
Do NOT punch
the tip through
the gel.
Dye spreading
under the well =
punctured well
K. Schramm
Proper Loading of
the Gel
Tip in buffer,
above well
Sample in well
K. Schramm
Return to Student Guide and
Complete Lab 1.2
Setting up the gel box
Remove tape before placing in the gel box
 Wells should be at the negative (black) end
 Buffer should just cover the gel – no dimples.
 Put the gel box in position before loading the
wells.
 Power supplies set at 135v
 Always turn power supply off before opening
gel box

Orange G (yellow), mol. Wt. = 452.4
Bromophenol blue (purple) = 691.9
Xylene cyanole (blue) = 538.6
K. Schramm
Lab 1.2 Results
A
3
2
1
Bromophenol blue
(purple) is more
negatively charged than
xylene cyanole (blue) due
to negatively charged
bromine ions. Therefore,
it travels farther than the
smaller xylene cyanole.