Transforming E. coli

• Thaw an eppendorf vial of competent cells on ice
• Mix the cells (between 20 and 200 ml) with DNA to be transformed.  The volume of the DNA should not exceed 1/10th of the volume of the competent cells.
• Incubate on ice for 30 min (shorter is possible, in fact, you can omit this step, although you efficiency will decrease).
• Place the cells in a waterbath maintained at 42 °C for 45 sec.  Cool down quickly on ice for about 2 minutes.
• Add medium (for best results use a rich medium like SOB, otherwise LC works fine) to 1 ml
• Incubate at 37 °C for 45 minutes
• Plate on selective plates.  Usually 100 ml works fine.  You may spin down the cells, resuspend the pellet and plate it, but do realize that this will lower the efficiency (with many selective markers plating 10 plates with 100 ml yields many more colonies than plating all cells on one plate).

Notes

Never refreeze thawed competent cells (or at the very least, never let others use them)

Whenever possible, add a negative control (no DNA, only the buffer you used to dissolve the DNA into) and a positive control (a plasmid that worked in the past, preferably a known amount).  Doing this every time will facilitate trouble shooting (and you are bound to be trouble shooting regularly)

Calculating the competence of your competent cells.   

Competence is expressed as colony forming units (cfu) per mg DNA.   It therefore varies with the specific DNA that your are using.  Often pUC18 is used as a 'standard'.  To compare different batches of competent cells, you need to use the same standard DNA.  Transform your cells with a known amount of DNA (for our standard cells 10 pg is enough).  Plate various volumes of your transformation (like 10, 40 and 200 ml).  Count the number of ensuing colonies on these plates and use these numbers to estimate how many colonies would have arisen had you plated the complete transformation mixture.  Use this number to calculate how many colonies would have arisen had you used 1 mg of DNA (in the case of 10 pg, multiply with 10⁵) 

Nico Stuurman, last updated 10-03-00