Post on 14-Jan-2016
transcript
JY BB JK JK CS LE MG AR
DS LL DH JB NG JS SJ YW MK
DNA Cloning
DNA Frag
Cloning Vector
Cloning Vectors - Plasmid
Other VectorsBacteriophageCosmidYAC
LigationT4 Ligase
GTCAGGTACCAGTC
CTGGCCA CATGGACCGGT
T4 Ligase (ATP)
GTCAGGTACCAGTC
CTGGCCA CATGGACCGGT
Sticky End LigationBlunt End Ligation
Ligation Reaction Mess
T4 Ligase ATP
Bacterial Transformation
Compentent E. coli
LB + Ampicillin
Dead
Colony
Dead
Dead
Dead
Colony
Dead
Colony
Selection
Screening Transformants
Insertional InactivationLacZ gene
Blue/Whiteccb gene
Dead/Alive
Step 1 Step 2
Size of InsertPCR + Electrophoresis
Miniprep + RE + Electro
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
Topo cloning vectors
Experimental Outline
PCR Reaction
TOPO Reaction
Bacterial Transformation
Miniprep + RE digest Electrophoresis
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
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°
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