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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