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