Team:Dundee/Team -




iGEM Sumbawagen 2014 · Econey

Notebook – Protocol – 8. Transformation (Heat Shock)

Protocols from the kit were followed by adaptation because we have no high speed micro centrifuge, but only a 6,000 rpm small micro centrifuge

1. Add plasmid into tubes containing E. coli competent cell.

2. Mix by pipetting using micropipette (5-10 times pipetting).

3. Incubate at 4 °C for 40 minutes (inside a refrigerator).

4. Prepare the hot water with the temperature of 42 °C, measure the temperature using thermometer.

5. Heat shock the cells by put the bottom of the tube on hot water for 45 seconds.

6. Incubate at 4 °C for 5 minutes.

7. Add 1 ml LB liquid medium sterile (without antibiotic) into each tubes.

8. Incubate using shaker for 2 hours

9. Plating the transformation by add 100 µl transformation reaction to LB agar plates containing chloramphenicol (final concentration 50 µg/ml).

10. Give label for the plates.

11. Centrifuge for 6,000 rpm for 3 minutes.

12. Discard the flow-through as much as 700 µl.

13. Suspend the remaining flow-through and pellets by pipetting.

14. Add 100 µl to agar plate containing appropriate antibiotic.

15. Give label for the plates.

16. Incubate at 37 °C overnight.

17. Check the result later.