Making competent E.coli is getting me desperate - After succeeding once, all tries after keep failing (Apr/27/2006 )
Hey,
I'm getting desperate about this. I have been trying to make competent cells for weeks now and apart from one time it always fails. I have gone through all the posts about the subject and tried several ways.
It must be something that i'm doing wrong but i have no idea what.
I prefer making electrocompetent cells but i've also tried making chemocompetent cells, both without succes. I'll give a description of what i alrady did:
electrocompetents :
auoclaved MQ, autoclaved 10% glycerol, ice cold recepients, even pipettes were ice cold.
I grew up a 5ml culture from a stock of top 10 e coli. Then took 2ml and grew them until OD600 = 0.4-0.6 in 200ml LB.
centrifuge at 6500g during 10 minutes in a precooled centrifuge (-5 - 0 °C)
resuspent pellet in 200 ml MQ
centrifuge at 6500g during 10 minutes in a precooled centrifuge (-5 - 0 °C)
resuspent pellet in 100 ml MQ
centrifuge at 6500g during 10 minutes in a precooled centrifuge (-5 - 0 °C)
resuspendt pellet in 4ml 10% glycerol
centrifuge at 6500g during 10 minutes in a precooled centrifuge (-5 - 0 °C)
resuspent pellet in 1ml glycerol and aliquot -80°C
This method gave me a good result once, after that it failed.
I also tried other methods, like using only 10% glycerol in the several steps.
chemocompetents :
I used the classic MgCL2 and CaCL2 method without succes.
I know making competent cells is a basic thing, i hope someone knows an answer.
thank you
Labkittie
Hello,
I will give a protocol for chemical competent bacteria.
It cannot fail.
Take 500 µL of overnight culture (16 hours) in 50 mL fresh medium. Let grow until OD = 0.3 (600 nm).
Put 30 mL of the bacteria on ice for 15 min.
Prepare fresh buffer : CaCl2 50 mM , Tris-HCl pH8. 10 mM. (make it fresh). There must be no precipitates, if there are : prepare new buffer again.
Filtrate it.
Centrifuge 5000g, 10 min, 4°C
Resuspend the pellet in 15 mL of CaCl2 buffer
Incubate 5 min on ice
Centrifuge 10 min at 5000g, 4°C
Discard supernatant.
Resuspend the bacteria in 1 mL of CaCl2 buffer.
For the transformation:
Mix 200 µL of bacteria with 50 ng of plasmide. Incubate 20 min on ice.
Incubate 90s at 42°C, cool the bacteria on ice few seconds (just to reach 37°C). Incubate 5 min at 37°C. Add 500 µL of LB. incubate 30-45 min at 37°C with shaking.
Inoculate an agar with LB and antibiotics. Let grow overnight.
Is there some differences with your protocol?
It's very important to centrifuge at 4°C.
It's very important to centrifuge at 4°C.
This is the protocol that i used to make chemocompetent cells.
3ml from an overnight culture(5ml) in 200ml LB medium --> until OD600 = 0.4-0.5 (i used 0.4)
transfer to an icecold centrifuge tube
centrifuge (@ 4°C) at 5000rpm for 10 minutes
dissolve pellet in 100ml 100mM MgCL2 pipetting up and down
5 minutes on ice
centrifuge at 4000 rpm for 10minutes (4°C)
carefully disolve the pellet in 10ml 100mM CaCL2
add the remaining 90ml CaCl2
20 minutes on ice
centrifuge at 4000rpm for 10 minutes at 4°C
resuspent pellet in 4ml icecold 85mMCaCL2+10% glycerol.
What do you use the Tris solution for?
to maintain pH8.0
OK, I don't know what could be bad in your protocol.
Oh, I forgot,
when I resuspend the pellet in CaCl2, I do it by swirling the tube (50 mL Falcon centrifuge tube) in the ice. it takes at least 5 minutes. I don't know if it's important.
I only did it this way, and it was working. I never tried to change anything.
I think the key is to avoid pitteting the cells up and down, because the competent cells are more fragile. I always use around 1000g when making competent cells. Since the pellet is not compact, after decanting the supernatant, sometimes I use pipette to remove trace supernatant. Then I can easily suspend the cells by adding cold sterilized water and swirling the centrifuge tubes (1 min).
Hope this would help.
Make SURE there is no detergent in the flasks used for culturing bacteria, and for the glassware used in handling the cells. A good way to do this is to autoclave the glassware with DI water, then dump it out.
Even very small amounts of detergent will make your competent cells fail, and this is an often overlooked factor in preparing cells.
pffff this is just impossible.
I tried all the suggestions and failed again. I am starting to think that there is something wrong with the cells rather then the method or the person executing it.
Is it necesairy to start from a invitrogen TOP10 vial? Now i've always started from a vial with e. coli that i had here...
do you pick one plated colony for culturing your cells?
it seems that your centrifuge is regulated in negatie temperatures?.. ( "-5 - 0 °C).
It's better to work at 4°
so plate a new vial of top 10 and pick one colony for preculture.
do you add AB in your culture?
it seems that your centrifuge is regulated in negatie temperatures?.. ( "-5 - 0 °C).
It's better to work at 4°
so plate a new vial of top 10 and pick one colony for preculture.
do you add AB in your culture?
The negative temps i dropped the last time and i indeed worked at 4°C. So i'll keep it that way.
I didn't plate out, i used 2µl of a glycerol stock that i had. Could that be it? Should i always plate out th bacteria?
I didn't add any AB.
I think temperature is critical for making competent cell. Keep it cold.
And before culture, restreak on plate is better to get the fresh cell for pre-culture.