AP Lab 6_ Molecular Genetics

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					AP Lab 6: Molecular Genetics
AP Lab 6 Day 1 Prelab

AP Lab 6: Part 1 Transformation
• • • • Restriction Enzymes Transformation Plating Transformation Efficiency

AP Lab 6: Part 1 Restriction Enzymes

5’ GAATTC 3’ CTTAAG

3’ 5’

5’ G AATTC 3’ 3’ CTTAA G 5’

AP Lab 6: Part 1 Restriction Enzymes

5’ GAATTC 3’ CTTAAG

3’ 5’

5’ G AATTC 3’ 3’ CTTAA G 5’

AP Lab 6: Part 1 Restriction Enzymes

AP Lab 6: Part 1 Restriction Enzymes

AP Lab 6: Part 1 Our plasmid
Rru I Xmn I Hind III Mst I Pvu I Bgl I ampr

-galactosidase
ori Pvu II Eco RI Bam HI Eco RI

AP Lab 6: Part 1 Transformation

• Bacteria are mixed with plasmids • Bacteria are “heat shocked” to encourage them to take in plasmids

AP Lab 6: Part 1 Plating

• Bacterial cells are suspended in a liquid. • The liquid is spread on the surface of a bacterial growth medium in a petri dish. • The liquid is spread to create a single layer of cells.

AP Lab 6: Part 1 Transformational Efficiency

• Bacterial colonies will start to grow on plates where they can metabolize the growth media.

Amp/X-GAL/pGAL

X-GAL/Control 1

Amp/X-GAL/Control 2

AP Lab 6: Part 1 Transformational Efficiency
Number of Transformants X g of DNA final volume Number of at recovery (mL) = Transformants volume plated (mL) per g

105

106

107

108

109

AP Lab 6: Part 1 Prelab Questions
• What is the purpose of the X-GAL/Control 1 plate? • What is the purpose of the AMP/XGAL/Control 2 plate? • What is the purpose of the AMP/XGAL/pGAL plate? • Which plate do you predict will have the highest transformation efficiency? Why?

AP Lab 6: Molecular Genetics
AP Lab 6 Day 2 Transformations

AP Lab 6 Part 1 Procedure Tips
• • • • Wash your hands with soap. Keep your work area clean. Label your tubes clearly. Unless otherwise directed, always keep the cells on ice. • Use sterile technique.

AP Lab 6: Part 2 Lab Overview

• • • • •

Restriction Enzymes Loading a gel Electrophoresis Staining a gel Interpreting results

AP Lab 6: Part 2 Restriction Enzymes

5’ GAATTC 3’ CTTAAG

3’ 5’

5’ G AATTC 3’ 3’ CTTAA G 5’

AP Lab 6: Part 2 Loading a Gel

• DNA sample is mixed with a dense, blue stain • Transfer pipets can be calibrated to small and precise volume measurements • Pipet tips will fit into the well • You should use two hands

AP Lab 6: Part 2 Electrophoresis

• Electrical current is applied to the sample • DNA fragments are polar • Different voltages and time lengths will results in quicker or clearer separations (not necessarily both)

AP Lab 6: Part 2 Staining a Gel

• After the gel has been run, the DNA is stained to make it easier to view • Methylene blue • Ethidium bromide

AP Lab 6: Part 2 Interpreting a Gel
Lane 1 Standard DNA Lane 2 Control DNA Lane 3 Patient Peripheral Blood DNA Lane 4 Patient Tumor DNA Lane 5 Patient Breast Normal DNA

AP Lab 6: Molecular Genetics
AP Lab 6 Day 3 Transformation Efficiency and Practice

AP Lab 6: Molecular Genetics
AP Lab 6 Day 4 Electrophoresis

AP Lab 6 Part 2 Procedure Tips
• Use two hands. • Double check you counting before you load a well. • Remember to avoid puncturing the gel! • Remember to change tips between samples. • Don’t eat the gel.

AP Lab 6: Part 2 Restriction Enzyme Cleavage of DNA & Electrophoresis
• LAB DAY – Load the gel with samples in lanes 2-6. – 1 = standard DNA fragments (Instructor) – 2 = student lab group sample – 3 = student lab group sample – 4 = student lab group sample – 5 = student lab group sample – 6 = student lab group sample 6

-

+
2

AP Lab 6 Part 2 Procedure Tips
• Thank your teacher for pouring your gel for you. • Consider coming in after school Wednesday to pour your own gel. • Thank your teacher for staining your sample for you after school. • Consider coming by after school Thursday to learn how to stain your sample.


				
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