Lab 3: Detecting an inserted gene

BIOSCI.101 Laboratory Course

This Course has six labs:

1. Enzymology 2. Gene Expression 3. Genetics 4 Blood Glucose 4. 5. Photosynthesis 6. Evolution

Reminder: BIOSCI 101 Incourse Test The test will be held on Thursday April 7th 6.30 8.30 pm (this week)
Information about test and room allocations are posted in Cecil and on the course notice board The test covers material from Cell & Molecular Biology , Microbiology and Genetics

IMPORTANT INFORMATION Take care when pouring and working with gels

The Schott bottle of agarose contains SYBR Safe. This binds with DNA and will fluoresce under UV light. (so you can see bands)

DANGER !
Equipment such as gel boxes and gel docs are used in other experiments and have residues of ethidium bromide. (This can causes mutations and cancer.)

Always wear gloves Al l when h handling h dli equipment with agarose Do not touch the agarose

The Rules!
Wear gloves when handling the Schott bottle or any equipment on the blue tray Remove gloves when handling other gear eg pencils, manuals etc (prevent contamination) Re-glove with a new pair when returning to add buffer and load DNA samples All used gloves go immediately into yellow BIOHAZARD BAGS not bins Always check with the tutor, demonstrators or technicians if you are unsure about anything

Gel Chamber
gel comb gel base

Seals at the end of the gel base gel chamber

Pouring an agarose gel (1)

Put on gloves before you start. Place casting tray SIDEWAYS into gel unit to f form a seal l at t the th sides id and d push h DOWN onto t the platform

SEALS

Pouring an agarose gel (2)

Position comb into slot

Pouring an agarose gel (3)

Once the gel chamber is ready, get a Schott bottle of agarose and GENTLY swirl the contents. contents

Pour all the liquid into the gel block.

Allow 15 minutes for the agarose gel to set.

Pipette reminder - You will be loading your gel with a P20

NAKED!

Pipette reminder - You will be loading your gel with a P20

TIP ON = READY TO GO!

While your gel sets..

Practise gel loading using perspex blocks with wells - BLUE DYE

Add 5ul of ORANGE loading dye to each of your 8 DNA samples l USING A NEW TIP each time TOTAL 15ul

Remember how to set the dial

Dial Reads eads 2.26l

Now that your gel is set

GENTLY pull the comb directly upwards Turn the casting tray around so that the wells are nearest to the black electrode (cathode)

Pour entire volume of buffer into the chamber. NOTE: the gel surface should be covered

Loading your samples into the gel

Use a new tip for each sample AND load them in order! (1, 2, 3, 4, 5, 6, +ve control, -ve control)
Top view Side view

Tips for gel loading

It helps if you support your arm Dont Don t put the tip all the way into the well, well just a little bit inside the well will be enough

DO NOT ATTACH THE GEL RIG TO THE POWER PACK YOURSELF WE WILL DO IT FOR YOU!

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Electrophoresis
DNA is -vely charged

Electrophoresis
Smaller fragments move faster through the gel than larger fragments

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Objectives of todays lab

1. Understand the PROCESS of detecting a foreign DNA s sequence including: i l di : - DNA extraction - How PCR works (including primer design!) - How electrophoresis works 2. Interpret an electrophoresis gel 3. Explain why +ve and -ve controls are required in PCR based experiments

CORN BORERS

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Corn borer control

Bacillus thuringiensis produces a
toxin that is lethal to certain insect larvae

Cry gene produces this toxic protein

ORGANIC FARM

Spray whole bacteria Plants transgenic for the Cry gene

NON-ORGANIC FARM

The scenario - page 3.10

FARMER JOE

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The process.
1 2 3 DNA extraction from organic g (?) ( ) corn PCR amplification Run a gel gel YOUR TASK

PCR
Our DNA of interest

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PCR

Heat to 95C DNA strands separate

PCR

Cool to 55C Primers anneal to DNA

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PCR

Heat to 72C Taq extends fragments

PCR

Heat to 95C DNA strands separate

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PCR

Cool to 55C Primers anneal to DNA

PCR

Heat to 72C Taq extends fragments

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How does PCR identify the region of DNA that you want amplified?
PCR is ULTRA specific due to the PRIMERS In your experiment where are the primers designed plant or bacteria DNA? (page 3.9) PLANT

So what does the result from hybrid corn look like?

Copy this slide and then make your own using this format format.

REMEMBER: Primers anneal to the plant DNA not the Cry gene

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In your experiment
Wild type Hybrid

Complexity! Why were the primers designed to the plant DNA and not the Cry gene?
Hybrid

Wild type
No PCR product

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When designed this way there is another possible source of the PCR product!

ORGANIC FARM NON-ORGANIC FARM

Spray whole bacteria Plants transgenic for the Cry gene

FALSE POSITIVE!!

The gel
wt = wild type H = Hybrid F = failed reaction
H F wt wt H wt +ve -ve

Think about WHY you need both positive AND negative controls!

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Clean up Instructions:
Leave buffer in gel boxes. DO NOT EMPTY. Place the following IN LABELLED CONTAINERS ON SIDE
a) perspex wells/combs b) microfuge tubes(i.e. gel sample tubes) c) tips (RETURN empty beaker to your ) yellow ll ti t b k t bench). d) agarose gel bottles

Empty ice bucket and stack on side bench.

FINAL CHECKLIST:
Assignment - name; stream number;
bench (#)

Completed Clean Up Wash hands before leaving

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XXXX MASTER SLIDE

Copy this slide and then make your own using this format.

XXXX MASTER SLIDE

Copy this slide and then make your own using this format.

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