Transformation of supercompetent cells
Inoculate two separate 1.5mls of LB medium with Invitrogen ( InvαF' ) cells .
Also include an uninoculated negative control tube .
Shake at 250 rpm overnight at 37˚C .
Inoculate with 500ul of overnight E . coli culture two 50ml tubes each containing 25mls of sterile SOB media .
Shake at 250 rpm for 4 hours .
Ice the tubes of cells for 15 mins .
Pellet cells ( approx 5 min spin , approx 3000 rpm ) and discard supernatant .
Resuspend each pellet of cells in 8 mls of cold TFB by washing gently , then sucking up and down , with a wide - bore 10ml pipette tip .
Ice the tubes of cells for 15 mins .
Pellet cells ( approx 5 min spin , approx 3000 rpm ) and discard supernatant .
Resuspend one pellet of cells in 2 mls of cold TFB and use this liquid to resuspend the second pellet .
You should now have all cells in a single aliquot of 2mls cold TFB .
Add 105ul of DMSO swirl and ice for 5 mins .
Add 80ul of 100mM DTT , swirl , and ice for 10 mins .
Add 105ul of DMSO swirl and ice for 5 mins .
Aliquot 180ul of cells into pre - chilled , labelled 5ml polypropylene growth tubes with loose - cap lids .
Include positive and negative controls .
Add ligation DNA to each sample ( in ~ 10ul volume ) and ice for 30 mins .
Heat pulse tubes at exactly 42˚C for exactly 90 secs .
Ice for 2 mins .
Add 400ul of 37˚C SOC to each tube and incubate at 37˚C for 10 mins .
Incubate a further 45 mins at 37˚C shaking at 225 rpm .
Place tubes in ice to halt growth .
Transfer the cells from each tube to a labelled individual 1.5ml microfuge tube .
Give the microfuge tubes a very short ( 5 sec ) spin to very loosely pellet the cells and then remove and discard 370ul of the supernatant .
Plate 40ul of the cell solution onto LB / Amp + / Xgal plates and grow overnight at 37˚C
