TITLE: Making electro-competent E. coli cells and transformation of them
AUTHORS: Jasper Koehorst

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes a method to make</span><span style = "font-style:italic;">Escherichia coli</span><span>cells electro-competent. The method involves one overnight culturing step, followed by several hours of culturing during the day and 1.5 - 2 hours of preparation of electro-competent cells.</span></div><div class = "text-block">The protocol also describes how to do the transformation (electroporation) afterwards.</div></div>

[STEPS]
?. [Day 2]
+ overnight cultureOriginal:Next day, first thing in the morning, inoculate 400 ml LB w/o salt in a 1 l Erlenmeyer flask with enough of the overnight culture to reach OD600 of 0.2 and incubate for ~3 hours to OD600 0.5-1.0.
[LB/ w/o salt in 1 Liter erlenmeyer flask]
[OD600]
?. [Day 2]
Original:Transfer culture volume to 8 Greiner tubes of 50 ml and cool on ice for 15 minutes.
[ice]
?. [Day 2]
Centrifuge for
[2000 g]
?. [Day 2]
Discard supernatant and addof , resuspend the pellet in it and then add  of , shake by hand a few times to mix.
5 ml
[glycerol]
45 ml
[glycerol]
?. [Day 2]
Centrifuge for 10 minutes at 2000g and 4°C.
?. [Day 2]
Discard supernatant and add 5ml of 10% glycerol, resuspend the pellet in it and then add 45ml of 10% glycerol, shake by hand a few times to mix.
?. [Day 2]
Put on ice for 10 minutes.
?. [Day 2]
Centrifuge for 10 minutes at 2000g and 4°C.
?. [Day 2]
Discard supernatant and resuspend the pellet in 1 ml ice-cold 10% glycerol, pool the contents of all 8 tubes into one tube.
?. [Day 2]
Centrifuge for 5 minutes at 1500g and 4°C.
?. [Day 2]
Discard supernatant and add 400 μl 10% glycerol (this will result in ~800 μl suspension).
?. [Day 2]
Make aliquots of 40μl in pre-cooled eppendorf tubes and flash freeze with N2(l). Store at -80°C.
?. [Day 1]
Inoculate  +  +  + Original:Inoculate 5 ml LB w/o saltin a 50 ml culturing tube withE. coliand incubate overnight at 37°C / 250 rpm
[LB w/o salt in 50ml tube]
[E.coli]
[Overnight]
?. 1.	Next day, first thing in the morning, inoculate 400 ml LB w/o salt in a 1 l Erlenmeyer flask with enough of the overnight culture to reach OD600 of 0.2 and incubate for ~3 hours to OD600 0.5-1.0.
?. 2.	Pre-cool centrifuge to 4°C.
?. 3.	Transfer culture volume to 8 Greiner tubes of 50 ml and cool on ice for 15 minutes.
?. 4.	Centrifuge for 10 minutes at 2000g and 4°C.
?. 5.	Discard supernatant and add 5ml of 10% glycerol, resuspend the pellet in it and then add 45ml of 10% glycerol, shake by hand a few times to mix.
?. 6.	Centrifuge for 10 minutes at 2000g and 4°C.
?. 7.	Discard supernatant and add 5ml of 10% glycerol, resuspend the pellet in it and then add 45ml of 10% glycerol, shake by hand a few times to mix.
?. 8.	Put on ice for 10 minutes.
?. 9.	Centrifuge for 10 minutes at 2000g and 4°C.
?. 10.	Discard supernatant and resuspend the pellet in 1 ml ice-cold 10% glycerol, pool the contents of all 8 tubes into one tube.
?. 11.	Centrifuge for 5 minutes at 1500g and 4°C.
?. 12.	Discard supernatant and add 400 μl 10% glycerol (this will result in ~800 μl suspension).
?. 13.	Make aliquots of 40μl in pre-cooled eppendorf tubes and flash freeze with N2(l). Store at -80°C.
?. [Day 2]
[OD600]
[OD600]