Team:UIUC-Illinois/Notebook/Protocols/Transformation

Select Protocol

• Bootcamp Protocols
• Subculturing Plates
• Making Electrocompetent E.Coli
• Making TAE Buffers
• Making Electrophoresis Gels
• Gel Purification
• Inoculation
• Miniprep
• PCR Protocols
• Digestions
• Ligations
• Transformation of E.Coli
• Storage of Cells

Electroporation and Transformation

You will need:

- 100 µl – Electrocompetent Cells
- 2-5 µl – Ligation Reaction (10 – 100 ng DNA)
- 1 ml – SOC broth/ LB liquid media
- Electrocuvette

Procedure:

1. Gather all your supplies and keep them on ice (or in 4o C room). This includes the cuvettes.
2. Thaw frozen electrocompetent cells on ice and place in electrocuvette. Then add ligation reaction and tap the cuvette to mix well. 3. DO NOT pipette up and down.
4. Keep on ice for 1 min.
5. Pulse once (On machine use Ec1 for 1mm cuvettes and Ec2 for 2mm cuvettes).
6. IMMEDIATELY after add 1ml of SOC broth and pipette up and down gently.
7. Incubate recovering cells in 37o C for 1 hr.

Plating:

1. After incubation, plate 200 µl of transformants on to appropriate selective plate and incubate at 37o C for 18+ hrs.
2. If no colonies are formed plate out the rest of the transformants (~800 µl) and incubate at 37o C for 24 hrs