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
