Team:CCU Taiwan/Project/Protocol

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Transform (homemade E-coli DH5α)

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).

2.Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.

3.Mix 5 μl of DNA into 50 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.

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

5.Heat shock each transformation tube into a 42°C water bath for 90 secs.

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

7.Add 50 μl LB to the bacteria and grow in 37°C shaking incubator for 180 min.

8.Spin down

9.Plate all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic (Chloramphenicol).

10.Incubate plates at 37°C overnight.