Bacterial Transformation

1. Take competent cells out of -80°C and thaw on ice. Pre-chill 1.5mL tubes in ice for every reaction mix.

2. Mix 1 μl of DNA (usually 10pg - 100ng) into 100 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.

-Do not pipet and GENTLY mix by flicking the bottom of the tube with your finger a few times.

-For transformation of ligated products, add 5 μl of ligation reaction mix.

3. Incubate the competent cell/DNA mixture on ice for 30 mins.

Cutting this incubation time short will slightly decrease transformation efficiency.

4. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 60 secs (45 secs is usually ideal)

In lab3, use the heat block, instead of the waterbath.

5. Put the tubes back on ice for 5 min.

6. Add 500 μl of LB or SOC medium.

7. Incubate cells for 1 hour at 37°C in a shaking incubator (200 - 250 rpm).

Place the eppendorf (1.5mL microcentrifuge) tubes into 50mL falcons (Max 3 per falcon). Take off the falcon's cap so there is good transfer of heat inside of it.

Ιncubation without shaking will decrease transformation efficiency.

Longer incubation of up to 2 hours can increase transformation efficiency.

During this step, remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let them warm up by incubating them (slightly open) for approximately 30 minutes in a 37°C incubator.

8. plating in LB-Cam plates:

- 50µL in one

- the rest 450µL in another

(for each sample)

9. OPTIONAL: Centrifuge at 8000 rpm for 3 minutes.

10. OPTIONAL: Remove 850µL of the supernatant.

11. OPTIONAL: Resuspend pelleted cells in the remaining LB or SOC medium.

12. Plate 100 μl onto a 10 cm LB agar plate containing the appropriate antibiotic.

13. Incubate plates at 37°C for 16-18 hours or overnight.

Do not forget to flip plates upside down after 30 minutes.

Calculating transformation efficiency

14. Efficiency (in cfu/µg) = [colonies on plate (cfu) / Amount of DNA plated (ng)] x 1000 (ng/µg)

15. Amount of DNA plated (ng) = Volume DNA added (1µL) x concentration of DNA (refer to vial, convert to ng/µL) x [volume plated (100µL) / total reaction volume (1000µL)]