2011 BIOSCI 101 Lab 3 Tutor template.pdf

8/14/2019 2011 BIOSCI 101 Lab 3 Tutor template.pdf

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Lab 3:

Detecting an

gene

BIOSCI.101 Laboratory Course

This Course has six labs :

1. Enzymology2. Gene Expression3. Genetics

.5. Photosynthesis6. Evolut ion


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Reminder: BIOSCI 101 Incourse Test

April 7 th

6.30 8.30 pm (this week)

Information about test and room allocations are postedin Cecil and on the course notice board

The test covers material from Cell & Molecular Biology ,Microbiology and Genetics

IMPORTANT INFORMATIONTake care when pouring and working

with els

The Schott bottle of agarose contains SYBRSafe .

This binds with DNA and will fluoresce underUV light. (so you can see bands)


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DANGER !Equipment such as gel boxes and gel docs areused in other experiments and have residues ofethidium bromide. (This can causes mutations

and cancer.)

ways wear g oves w en an ng

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 egpencils, manuals etc (prevent contamination)

Re-glove with a new pair when returning to addbuffer and load DNA samples

BIOHAZARD BAGS not b ins

Always check with the tuto r, demonstrators ortechnicians if you are unsure about anything


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Gel Chamber

gel comb

Seals at the endof the gel base

gel chamber

Pouring an agarose gel (1) Put on gloves before you start.

Place casting tray into gel unit toSIDEWAYSorm a sea a e s es an pus on o

the platform

SEALS


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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.

Pour all the liquid into the gel block.

Allow 15 minutes for the agarose gel to set.


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Pipette reminder - You will beloading your gel with a P20

NAKED!

Pipette reminder - You will beloading your gel with a P20

TIP ON =READY TO GO!


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While your gel sets..

with wells - BLUE DYE

Add 5ul of ORANGE loading dye to each ofyour samp es

each time TOTAL 15ul

Remember how to setthe dial

DialReads2.26 l


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Now that your gel is set GENTLY pull the comb directly upwards

Turn the casting tray around so that the wellsare nearest to the black electrode cathode

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


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Loading your samples into the gel

Use a new tip for each sample AND load themin order! (1, 2, 3, 4, 5, 6, +ve control, -vecontrol)

Top view Side view

Tips for gel loading It helps if you support your arm

,little bit inside the well will be enough


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DO NOT ATTACH THE GEL RIG TO THEPOWER PACK YOURSELF

WE WILL DO IT FOR YOU!


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Electrophoresis

DNA is -vely charged

Electrophoresis Smaller fragments move faster through the


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Objectives of todays lab

1. Understand the PROCESS of detecting a foreign DNAsequence nc u ng:

- 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

insect larvae

Cry gene produces this toxic protein

NON-ORGANIC FARM Plants transgenicfor the Cry

gene

The scenario - page 3.10

FARMER JOE


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The process.

DNA extraction from or anic (?) corn1

PCR amplification2

PCROur DNA of interest


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PCR

Heat to 95 C

DNA strands separate

PCR

Cool to 55 C

Primers anneal to DNA


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PCR

Heat to 72 C

Taq extends fragments

PCR

Heat to 95 C

DNA strands separate


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PCR

Cool to 55 C

Primers anneal to DNA

PCR

Heat to 72 C

Taq extends fragments


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How does PCR identify the region ofDNA 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 cornlook like?

Copy this slide and then make your own using.

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 PCRproduct


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

ORGANIC FARM Spray whole bacteria

NON-ORGANICFARM

Plants transgenicfor the Cry gene

FALSE POSITIVE!!

The gel

H F wt wt H wt +ve -veH = Hybrid

wt = wild type

F = failed reaction

Think about WHY you need both positive ANDnegative controls!


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XXXX MASTER SLIDE

using this format.

XXXX MASTER SLIDE

using this format.

2011 BIOSCI 101 Lab 3 Tutor template.pdf

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