Transforming a plasmid

This is a basic transformation protocol you can use when you just need to make a lot more of an already constructed plasmid.


 * 2uL 250ng/uL DNA + 1mL H2O ( ~0.5ng/uL). This doesn’t have to be exact…


 * Pre-chill 2uL of this dilution in an eppendorf (ie: transform with around 1ng plasmid)


 * Turn water bath on to 42C


 * Thaw DH5a (or other) cells on ice ~10 mins; flick to re-suspend (dont pipette! this will damage the bugs)
 * ​These are located red boxes on third row from bottom in -80 freezer by cryostat. (100ul aliquots)


 * Clean your bench with 70% EtOH


 * Dry LB-amp plates by propping open 10 mins on clean bench
 * ​Best to do this upside down (so the agar side is not face up)


 * Add 45uL re-suspended cells to 2uL pre-chilled dilution
 * ​Volume of cells used depends on how good the cells are (ie: top10 from biostores, use 15-20uL instead of 45uL)


 * Set cells on ice 20 -45 minutes // preheat plates 20mins in 37C incubator


 * Heat shock cells 30sec at 42C in water bath


 * Return cells to ice


 * Plate (use sterile glass beads)
 * ​5-6 beads/plate, rock back and forth (dont swirl) until bacteria are evenly plated


 * Set at 37C O/N (agar side up)