Program IntroductionProgram IntroductionChapter 1 – Some Tools of the TradeChapter 1 – Some Tools of the Trade
Key ideas: IntroductionKey ideas: IntroductionGenetic 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 ideasKey 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 takeThe Steps we will take 1.Learn to use the equipment
1. micropipeting & gel electrophoresis
2.Prepare a plasmid to insert into bacterium3.Verify the uptake of gene4.Transforming bacteria with recombinant plasmid
Skills & labs for this unitSkills & labs for this unit
Using equipmentPractice setting volumes on the pipettesAloquoting with pipettesLoading, running & reading gelsAdding gene fragments to plasmidUse enzymes to cut and splice gene fragmentsVerifying gene uptakeUse gel electrophoresis to verify plasmid has gene fragmentTransforming E. Coli with a recombinant plasmidTransformation & verification on growth plates
Video 1Video 1 Video 2Video 2
Using the Micropipette
Types of MicropipettesTypes of MicropipettesLockLock
Pipetting TechniquePipetting Technique
Hold the micropipette and microfuge tubes at eye level when loading or dispensing samples
TipsTips
If you are pipetting, you should be holding the tube.
Common mistake!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 Loading Gels
Insert pipette tip:•Under buffer level•Above gel well
Different pipetting techniques – stability is the keyDifferent pipetting techniques – stability is the key
K. Schramm
K. SchrammK. Schramm
Tip should be above, not in the well.
Do NOT punch the tip through the gel.
Dye spreading under the well = punctured well
Common Loading errors
Proper Loading of the Gel
Tip in buffer, above well
Sample in well
K. Schramm
Return to Student Guide and Complete Lab 1.2
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
Setting up the gel box
Lab 1.2 Results Lab 1.2 Results K. Schramm
Orange G (yellow), mol. Wt. = 452.4
Bromophenol blue (purple) = 691.9
Xylene cyanole (blue) = 538.6
AA
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.
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