Competent Cells

From FreeBio

{The following protocol, modified from Short Protocols in Molecular Biology, 4th Ed., is used to make cells chemically competent for transformation. Remember to use sterile techniques, since there is no selective marker}

Making Cells Competent

1. Inoculate a single colony into 5ml LB medium. Grow overnight @ 37°C on shaker.
2. Inoculate 4ml of culture into 400ml LB medium in 2l flask. Grow @ 37°C on shaker to OD₅₉₀ of 0.375.
   {400ml culture makes 16ml of competent cells, or 64 tubes of 250µl aliquots. Use less volume if fewer cells are needed, and adjust protocol as appropriate.}
   • Make CaCl₂ soln and keep on ice.
   • Chill eight 50ml polypropylene tubes on ice.
   • Turn down centrifuge (in Rm 5086) thermostat to 4°C.
3. Aliquot culture into eight 50-ml tubes, and incubate on ice for 5-10min. Spin down cells for 7' @ 1600xg, 4°C.
4. Gently resuspend each pellet in 10ml ice-cold CaCl₂ soln. Spin down cells for 5' @ 1100xg, 4°C.
5. Resuspend each pellet in 10ml cold CaCl₂ soln. Incubate cell suspension on ice for 30'.
   • Chill seventy 1.5ml cryotubes on ice.
   • Chill storage box on ice.
6. Spin down cells for 5' @ 1100xg, 4°C.
7. Resuspend each pellet competely in 2ml ice-cold CaCl₂ soln.
8. Aliquot 250µl into pre-chilled 1.5ml tubes. Freeze @ -80°C.

Transforming w. Competent Cells

1. Add 10 ng DNA (5-25 µl) into 1.5ml tube. Place on ice.
2. Rapidly thaw competent cells by warming between hands, dispense 50µl immediately into test tubes containing DNA. Swirl and incubate on ice for 10'.
3. Heat shock cells for 2' @ 42°C.
4. Add 500µl LB (or SOC) medium to each tube. Incubate 1h @ 37°C on shaker.
5. Plate (all) on selective medium and incubate overnight @ 37°C.

Posted by dna1112 13:04, 11 Jul 2005 (EDT)