MIT iGEM Top10 ChemComp Ecoli transformation protocol

Regular Transformation protocol for Top10 cells

• Take out an appropriate aliquot of Top10 ChemComp cells from -80 freezer (Very top right rack on top shelf, bottom slide-out shelf).
  • Let cells thaw ON ICE! for ~5-10min. Cells MUST be kept cold until heat shock! This is critical!
  • Aliquots are either 50µl (small PCR tubes) or 200µl (bigger tubes). If you're transforming multiple (>=4) DNA samples, aliquot 50µl into PCR tubes from the 200µl aliquot.
• Transform 50 μl of cells with 3-4µl of ligated DNA
• Keep ON ICE 30min. This step is to let the salt from the ligation equilibrate over the cells.
• Heat shock 60 sec at 42C. You can set a thermocycler to 42*C instead of making a water bath.
• Put cells back on ice for 2min.
• Add your 50µl of cells to 200 μl SOC in a 2ml epp tube.
• Incubate at 37 C for 1 hour. You can just place the 2ml epp tubes in a little plastic cup and put them on the shaker in the 37*C room.
  • Using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
  • For plasmids pSB1AC3 and pSB1AT3, which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies. This might be because the cells double in that time or it might be because more Ab-resistance protein is made.
  • Ampicillin and kanamycin appear to do fine with 1 hour growth
• Plate 200 μl (or less if you're expecting a ton of transformants) on the appropriate antibiotic plates. Use either hockey stick in ethanol or sterilized glass beads to spread.

To Test Competence:

• Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
  • This is at 10 pg/μl or 10^-5 μg/μl
  • This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE
• Hold on ice 0.5 hours
• Heat shock 60 sec at 42C
• Add 250 μl SOC
• Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
  • using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
  • For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
  • Ampicillin and kanamycin appear to do fine with 1 hour growth
• Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
• Transformation efficiency is 15 x colony count x 10⁵
  • We expect that the transformation efficiency should be between 5x10⁸ and 5x10⁹