I have a limited stock of Stbl3 chemically competent cells and would like to grow a stock that can be used for future experiments. Could some one recommend a protocol for growing chemically competent cells?
Hi Mr Potts,
We prepare Top10 E-coli competent cells by rubidium as follows:
Preparation of competent cells:
- Inoculate 100 ml of Psi medium with 1 ml of a preculture Top 10;
- Place the culture at 37 ° C with stirring until an OD 550 of about 0.4 to 0.5;
- Cool the Erlenmeyer flask in ice for 15 min;
- Cold Centrifuge for 5 min at 5000 rpm in falcons cooled ;
- At the pellet, add 40 ml of buffer TFBI and icubate 15 min on ice;
- Centrifuge 5 min at 5000 rpm;
- Remove the supernatant and resuspend the pellet in 4 ml of buffer TfbII;
- Keep the rest in 200 .µl aliquots at-70° C.
Transformation with a plasmid:
- At 100µl competent cells, add 2-4 .µl of the ligation product and incubate for 30 min on ice;
- Perform a heat shock at 42 ° C for 2 min followed by 2 min in ice;
- Add 1 ml LB and incubated at 37 ° C for 1 hour (without stirring);
- spread on solid mediumand incubated overnight at 37 ° C.
Buffer:
Tampon TFbI : (pH 5,8)
- Acétate de potassium : 30 mM ;
- RbCl : 100 mM ;
- CaCl2 : 10 mM ;
- MnCl2 : 50 mM ;
- Glycerol 15% (V/V).
Tampon TFbII: (pH 6,5 )
- MOPS: 10 mM ;
- RbCl: 10 mM ;
- CaCl2: 75 mM ;
- Glycerol 15% (V/V).
Good luck
Just streak on LB Agar with desired antibiotic. These dont need particular protocol to grow. From plates grow in liquid culture for chmeical competent cell preparation.
Hi Mr Potts,
We prepare Top10 E-coli competent cells by rubidium as follows:
Preparation of competent cells:
- Inoculate 100 ml of Psi medium with 1 ml of a preculture Top 10;
- Place the culture at 37 ° C with stirring until an OD 550 of about 0.4 to 0.5;
- Cool the Erlenmeyer flask in ice for 15 min;
- Cold Centrifuge for 5 min at 5000 rpm in falcons cooled ;
- At the pellet, add 40 ml of buffer TFBI and icubate 15 min on ice;
- Centrifuge 5 min at 5000 rpm;
- Remove the supernatant and resuspend the pellet in 4 ml of buffer TfbII;
- Keep the rest in 200 .µl aliquots at-70° C.
Transformation with a plasmid:
- At 100µl competent cells, add 2-4 .µl of the ligation product and incubate for 30 min on ice;
- Perform a heat shock at 42 ° C for 2 min followed by 2 min in ice;
- Add 1 ml LB and incubated at 37 ° C for 1 hour (without stirring);
- spread on solid mediumand incubated overnight at 37 ° C.
Buffer:
Tampon TFbI : (pH 5,8)
- Acétate de potassium : 30 mM ;
- RbCl : 100 mM ;
- CaCl2 : 10 mM ;
- MnCl2 : 50 mM ;
- Glycerol 15% (V/V).
Tampon TFbII: (pH 6,5 )
- MOPS: 10 mM ;
- RbCl: 10 mM ;
- CaCl2: 75 mM ;
- Glycerol 15% (V/V).
Good luck
Phani kashyap is right. Yo can not mantain the competence of your cells. You can freeze your strain but before to use it un a transformation experiment you have to make it competent again
try this protocol it is very good.
http://www.molbi.de/protocols/competent_cells_chemical_v1_0.htm
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