JY BB JK JKCS LE MG AR DS LL DH JB NG JS SJ YW MK.
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Transcript of JY BB JK JKCS LE MG AR DS LL DH JB NG JS SJ YW MK.
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JY BB JK JK CS LE MG AR
DS LL DH JB NG JS SJ YW MK
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DNA Cloning
DNA Frag
Cloning Vector
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Cloning Vectors - Plasmid
Other VectorsBacteriophageCosmidYAC
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LigationT4 Ligase
GTCAGGTACCAGTC
CTGGCCA CATGGACCGGT
T4 Ligase (ATP)
GTCAGGTACCAGTC
CTGGCCA CATGGACCGGT
Sticky End LigationBlunt End Ligation
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Ligation Reaction Mess
T4 Ligase ATP
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Bacterial Transformation
Compentent E. coli
LB + Ampicillin
Dead
Colony
Dead
Dead
Dead
Colony
Dead
Colony
Selection
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Screening Transformants
Insertional InactivationLacZ gene
Blue/Whiteccb gene
Dead/Alive
Step 1 Step 2
Size of InsertPCR + Electrophoresis
Miniprep + RE + Electro
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PCR Cloning
• Taq Polymerase ends
• Adds extra Adenosine onto 3’ end– Generating a 3’ A overhang
AA
PCR Product
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
1. Mix• 4 μl PCR reaction• 1 μl Salt Solution• 1 μl Topo vector
2. Incubate 5 min at room temperature
3. Set up Transformation
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Transformation
1. 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.
2. 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.
3. Heat shock cells for 30 seconds at 42° - immediately return to ice.
4. Add 250 μl room temp SOC media – cap tube and shake horizontally for 1 hour
5. Spread 10, 50, 100 μl of cells on prewarmed LB+amp plates.
6. 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• Miniprep Isolation of DNA• Restriction Digest• Gel Electronphoresis