Invirtrogen cloning

Cloning – Invitrogen TOPO-TA kit

Ligation

1. Add to the reaction mixture:

4 µl Fresh PCR product

1 µl Salt Solution

1 µl TOPO vector

2. Incubate at room temperature for 5 minutes

3. Store on ice until needed, or overnight at 4°C for maximum number of transformants

Transformation

1. Get cells out of freezer, thaw on ice for 5 minutes

2. Add 2 µl of ligation mixture

3. Incubate on ice for 5 minutes

4. Heat-shock the cells for 30 seconds at 42°C

5. Immediately put the cells on ice

6. Add 250 µl of SOC recovery media

7. Shake at 37°C for 1 hour

8. Spread 50 µl on LB plates with X-gal and ampicillin

9. Incubate plates overnight at 37°C and you should get colonies

10. Proceed to colony PCR to verify your results

Materials necessary

Cleaned PCR product

10 µl pipette tips

1000 µl pipette tips

1.5 ml microcentrifuge tube

Ice

Equipment necessary

37° C shaking incubator

37° C incubator

10 µl micropipette

1000 µl micropipette

42° C heat block