AP Lab 6: Molecular Genetics

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AP Lab 6 Molecular Genetics

• AP Lab 6 Day 1
• Prelab

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AP Lab 6 Part 1 Transformation

• Restriction Enzymes
• Transformation
• Plating
• Transformation Efficiency

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AP Lab 6 Part 1 Restriction Enzymes

• 5 GAATTC 3 5 G AATTC 3
• 3 CTTAAG 5 3 CTTAA G 5

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AP Lab 6 Part 1 Restriction Enzymes

• 5 GAATTC 3 5 G AATTC 3
• 3 CTTAAG 5 3 CTTAA G 5

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AP Lab 6 Part 1 Restriction Enzymes
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AP Lab 6 Part 1 Restriction Enzymes
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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

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AP Lab 6 Part 1 Transformation

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

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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.

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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
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AP Lab 6 Part 1 Transformational Efficiency

• Number of final volume Number of
• Transformants X at recovery (mL)
  Transformants
• ?g of DNA volume plated (mL) per ?g
• 105 106 107 108 109

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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/X-GAL/Control 2
  plate?
• What is the purpose of the AMP/X-GAL/pGAL plate?
• Which plate do you predict will have the highest
  transformation efficiency? Why?

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AP Lab 6 Molecular Genetics

• AP Lab 6 Day 2
• Transformations

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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.

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AP Lab 6 Part 2Lab Overview

• Restriction Enzymes
• Loading a gel
• Electrophoresis
• Staining a gel
• Interpreting results

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AP Lab 6 Part 2 Restriction Enzymes

• 5 GAATTC 3 5 G AATTC 3
• 3 CTTAAG 5 3 CTTAA G 5

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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

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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)

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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

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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

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AP Lab 6 Molecular Genetics

• AP Lab 6 Day 3
• Transformation Efficiency and Practice

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AP Lab 6 Molecular Genetics

• AP Lab 6 Day 4
• Electrophoresis

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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.
• Dont eat the gel.

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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

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2
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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.