Protocol Electro-transformation : Select a snigle colony of E.coli from fresh LB plate and inoculate to 10 ml LB broth medium.Incubate until to reach log phase. Inoculate 330 ml of LB media with 10 ml starter culture and grow in 37 shaker. When the OD600 reaches 1,aliquot it in 50 ml tubes and immediately put the tubes on ice. Chill it for minutes. Pellet the bacteria by centrifugation(3300 rpm,7min, 4°C) Resuspend cell pellet per tube in 4 ml of Hepes 1mM (ph~ 7) Incubate min Pellet the bacteria by centrifugation Remove supernatant by inverting the tubes.Resuspend pellet per tube in 10 ml glycerol, Incubate on ice for 20 min Pellet the bacteria by centrifugation. Remove supernatant.
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Protocol Electro-transformation : Select a snigle colony of
E.coli from fresh LB plate and inoculate to 10 ml LB broth
medium.Incubate until to reach log phase. Inoculate 330 ml of LB
media with 10 ml starter culture and grow in 37 shaker. When the
OD600 reaches 1,aliquot it in 50 ml tubes and immediately put the
tubes on ice. Chill it for minutes. Pellet the bacteria by
centrifugation(3300 rpm,7min, 4C) Resuspend cell pellet per tube in
4 ml of Hepes 1mM (ph~ 7) Incubate min Pellet the bacteria by
centrifugation Remove supernatant by inverting the tubes.Resuspend
pellet per tube in 10 ml glycerol, Incubate on ice for 20 min
Pellet the bacteria by centrifugation. Remove supernatant.
electroporator Elecrotransformat ion 2 Comments: 1.Purify the
ligation product 2.Desault medium with mili Q water 3.New cuvett,
size cuvett 4.Fresh bacteria 5.Cold room 6.Determine the voltage,
capacity, resistancy Clean the ligation as follow : 1.Heat the
reaction at 65C for 10 min 2.Add phenol,vortex, centrifuge at
13000rpm,10 min.Transfer upper phase to a fresh microtube. 3.Add to
the upper phase chloroform,vortex,centrifuge at 13000rpm,10 min.
4.Add to upper phase NaAcetate 3M, 5.Add ethanol, mix, 6.Store -70
for at least 30 min 7.Centrifuge at 13000rpm,20 min,remove
supernatant 8.Dissolve pellet in H2O.
