pGEM cloning

Cloning - Promega pGEM-T Easy kit

Ligation

1. Add to the reaction mixture:

a. 3 µl Fresh PCR product

b. 5 µl 2x Rapid Ligation Buffer

c. 1 µl pGEM-T vector

d. 1 µl T4 DNA Ligase

2. Incubate at room temperature for 1 hour or overnight at 4°C

3. Store on ice until needed

Transformation

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

2. Add 2 µl of ligation mixture into a 1.5 ml tube on ice

3. Carefully transfer 50 µl of cells into the tube

4. Incubate on ice for 20 minutes

5. Heat-shock the cells for 45-50 seconds at 42°C

6. Immediately put the cells on ice for 2 minutes to recover

7. Add 950 µl of SOC recovery media

8. Shake at 37°C for 1.5 hours

9. Spread 100 µl onto LB plates with X-gal and ampicillin

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

Materials required

Cleaned PCR product

10 µl, 200 µl, and 1000 µl pipette tips

1.5 ml microcentrifuge tubes

Ice

Equipment necessary

10 µl micropipette

200 µl micropipette

1000 µl micropipette

37° C incubator

37° C shaking incubator

42° C heat block