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 &mu;l 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 &mu;l 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 &mu;l (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 &mu;l of cells with 1 &mu;l of standard pUC19 plasmid (Invitrogen)
 * This is at 10 pg/&mu;l or 10-5 &mu;g/&mu;l
 * This can be made by diluting 1 &mu;l of NEB pUC19 plasmid (1 &mu;g/&mu;l, NEB part number N3401S) into 100 ml of TE
 * Hold on ice 0.5 hours
 * Heat shock 60 sec at 42C
 * Add 250 &mu;l 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 &mu;l 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