JY BB JK JK

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JY BB JK JK
CS LE MG AR
NG JS SJ YW MK
DS LL DH JB
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DNA Cloning
DNA Frag
Cloning Vector
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Cloning Vectors - Plasmid
Other Vectors Bacteriophage Cosmid YAC
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LigationT4 Ligase
CTGGCCA CATGGACCGGT
GTCAGGTAC CAGTC
T4 Ligase (ATP)
GTCAGGTAC CAGTC
CTGGCCA CATGGACCGGT
Sticky End Ligation Blunt End Ligation
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Ligation Reaction Mess
T4 Ligase ATP
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Bacterial Transformation
Dead Colony Dead Dead Dead Colony Dead C
olony
Selection
LB Ampicillin
Compentent E. coli
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Screening Transformants
Step 1
Step 2
Insertional Inactivation LacZ gene Blue/White
ccb gene Dead/Alive
Size of Insert PCR Electrophoresis Miniprep
RE Electro
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PCR Cloning

• Taq Polymerase ends
• Adds extra Adenosine onto 3 end
• Generating a 3 A overhang

A
PCR Product
A
TA cloning vectors Ligase Topo cloning vectors
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Topo cloning vectors
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Experimental Outline
PCR Reaction TOPO Reaction Bacterial
Transformation Miniprep RE digest
Electrophoresis
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Topo Reaction

• Mix
• 4 µl PCR reaction
• 1 µl Salt Solution
• 1 µl Topo vector
• Incubate 5 min at room temperature
• Set up Transformation

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Transformation

• Gently thaw TOP10 cells on ice It is essential
  to keep cells on ice at all times. Even a few
  seconds at room temperature can ruin experiment.
• Add 2 µl Topo reaction to cells and incubate on
  ice for 15 minutes. mix gently by stirring with
  pipette tip, do not pipette up and down.
• Heat shock cells for 30 seconds at 42 -
  immediately return to ice.
• Add 250 µl room temp SOC media cap tube and
  shake horizontally for 1 hour
• Spread 10, 50, 100 µl of cells on prewarmed
  LBamp plates.
• Grow O/N at 37

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Experimental Time Table

• Wednesday 9/27
• Lecture on Restriction Enzymes
• Thursday 9/28
• Topo Cloning Reaction
• Transformation
• Restriction analysis of Fragment
• Solutions for DNA Isolation
• Friday 9/29
• Check and refrigerate transformants
• Monday 10/2
• Lecture on DNA Isolation
• Wednesday 10/4
• Pick colonies streak and start O/N cultures
• Thursday