1. Background and purpose of the procedure
Transformation is a key process in molecular cloning, by which multiple copies of recombinant DNA molecules are produced. The ability to take up free, extracellular genetic material is the prerequisite for bacterial competent cells to undergo transformation. The objective is to obtain the replication of sequence of interest of a recombinant plasmid.
2. Materials and equipment
- Chemically competent DH5α bacteria (Invitrogen, 18265017)
- pDNA to be amplified
- 2xYT broth (see 3. Recipes)
- Appropriate selection antibiotic (see 3. Recipes)
- Ice bucket
- Heated water bath
- Shaker incubator
- Bunsen burner for sterile experimentation space
- 250 mL sterile Erlenmeyer for liquid culture (preferably baffled)
- 14 mL sterile polypropylene culture tubes (Corning Falcon, 352059)
31 g/L of 2X Yeat Extract Tryptone (BD Difco, 244020) [62 g for 2 L]
ddH2O qsp 2L
Adjust pH to 7.0 and sterilize using autoclave at 121˚C for 15 min minimum.
Solution can be stored at room temperature for several months without antibiotics.
Carbenicillin 1000x stock solution (for ampicillin resistant pDNA)
100 mg/mL Carbenicillin disodium (GoldBio, C-103) [1 g for 10 mL]
50% Ethanol in ddH2O qsp 10mL
Sterilize through 0.22µm filters, aliquot stock solution in 1.5 mL microtubes and store at -20˚C until use. Solution can be stored at -20˚C for up to 12 months and is stable for few weeks at 4˚C.
Kanamycin 1000x stock solution
50 mg/mL Kanamycin monosulfate (GoldBio, K-120) [0.5 g for 10 mL]
ddH2O qsp 10 mL
Sterilize through 0.22µm filters, aliquot stock solution in 1.5 mL microtubes and store at -20˚C until use. Solution can be stored at -20˚C for up to 12 months and is stable for few days at 4˚C.
- Allow competent bacteria to thaw on ice, gently flick the tube multiple times to obtain a uniform suspension
- Under sterile conditions (use of the Bunsen burner), transfer 50 µL of competent bacteria to a pre-chilled 14 mL sterile polypropylene culture tube
- Add 1 to 100 ng of pDNA to be amplified to the bacteria and gently flick the tube to ensure proper mixing
- Incubate on ice for 10 to30 min
- Transfer the bacteria to the water bath pre-heated at 42˚C for 42 seconds
- Transfer the bacteria to ice for 5-10 min
- Add 250 µL of 2xYT broth to the bacteria
- Incubate the bacteria in a shaker incubator at 37˚C for 1 hour
- Pipet 10 to 100 µL of the transformed cell suspension and inoculate 50 mL of 2xYT containing the appropriate selection antibiotic (Carbenicillin or Kanamycin)
- Incubate in a shaker incubator at 37˚C for 16 hours (overnight)
Alternatively, bacteria can be spread on top of a 2xYT agar plate containing appropriate selection antibiotic for colony picking.
Molecular Cloning: a Laboratory Manual (3rd Edition), by J. Sambrook and D.W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001.
6. Revision history
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|Summary of modifications|
|Initial version of the protocol|
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