How do I make more competent cells? - by growing up and freezing glycerol stock? (Jun/26/2005 )
hii, i use MgCl2-Cacl2(80mm- 20mm )15 ml for 50 ml culture media,
then .1 m cacl2 10 ml,
then dissolve the comp cell in the 2 ml of cacl2.
and i always make the comp cell fresh, and some time i prepare 1 day before the transformtion.
and i get very good results. a very good no of transformnts.
i refer the "Molecular cloning" by Sambrook and Russell , new edtion.
hi,
i usually keep colonies by growing them in LB broth and then store them with glycerol in -80oC for more than a year. so the next time you want to use it, just plate the culture on plate in zigzag fashion directly to gain single colony and grow them up. it saves you from doing transformation all the time.
i use the protcol of the "molecular cloning". by Sambrook and Russsell".
the method of my choice is CaCl2 method.
it is very effective method.
i use the protcol of the "molecular cloning". by Sambrook and Russsell".
the method of my choice is CaCl2 method.
it is very effective method.
Hi everyone.
I use also to do a lot of cloning. I prepare a stock of competent cells using calcium chloride technique. I keep it at -80 degrees. After use, the left cells I always throw or grow them to make a new stock.
competent cells are no more able to uptake the DNA after growing them, you have to make them competent again by fragilizing the membrane by the calcium then keep them at very cold temperatute -80 degree until use.
I can keep my cells up to one year they are sstill ok.
i use regulary electrocompetent cells. to make a new stock i plate them on selective plate, do a preculture (w selection) and a bigger culture (w/o selection). then stop the culture on ice 30', pellet them, make 3 washes and a wash in 10% glycerol. Finally resuspend the cells in small volume of 10% glycerol and freeze them in liquid nitrogen and then store at -80°. Hence i assume they are ready to use because i just thaw them on ice and add DNA before electroporation.
So i assume they've been made competent by the 10%glycerol wash?
Fred
Ps : if you want the detailed protocol i use, i can mail it to you.
Hi fred, thanks for that. I'll like to go one step back to ask you about chemical vs electrocompetent cells. I have a batch of BL21DE3 from Invititrogen, and thought of making more for the future. According to Invitrogen, these cells are chemically competent, not electro. Can you tell me what makes cells suitable for one type of transformation method and not the other? Can your electroporation competency protocol be applied to my BL21DE3 to make it electrocompetent, or is it that once it is chem competent, it cannot be converted to electrocompetent?
hi
according to the person who have prepared chemically competent cells in my lab, two things :
1 : protocols are different, and i asked her if i can get the protocol
2 : seems that electrocompetent and chemically cometent cells can not cross over the line cehm/electro...
i use regulary electrocompetent cells. to make a new stock i plate them on selective plate, do a preculture (w selection) and a bigger culture (w/o selection). then stop the culture on ice 30', pellet them, make 3 washes and a wash in 10% glycerol. Finally resuspend the cells in small volume of 10% glycerol and freeze them in liquid nitrogen and then store at -80°. Hence i assume they are ready to use because i just thaw them on ice and add DNA before electroporation.
So i assume they've been made competent by the 10%glycerol wash?
Fred
Ps : if you want the detailed protocol i use, i can mail it to you.
Hi fred, thanks for that. I'll like to go one step back to ask you about chemical vs electrocompetent cells. I have a batch of BL21DE3 from Invititrogen, and thought of making more for the future. According to Invitrogen, these cells are chemically competent, not electro. Can you tell me what makes cells suitable for one type of transformation method and not the other? Can your electroporation competency protocol be applied to my BL21DE3 to make it electrocompetent, or is it that once it is chem competent, it cannot be converted to electrocompetent?
Hi. You do need to make these cells competent before they will work. I use a method using chem compatency. This is usually achieved by growing at 18 degrees(which takes along time) in SOB medium and making competent by using a specially made buffer. This paper describes a very good (but long winded) method which is very good for long term storage of these competent cells = Chung, C. T. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172-5. This method is very reproducible for preparing a variety of different strains of E. Coli. There is a quicker method but these cells do not retain their competency when frozen at -80. The paper that describes this method is Inoue et al. Gene. 1990 Nov 30;96(1):23-8.
Hope this helps.
I always prepare fresh competent cells using CaCl2. Never try keeping them in -80 and use some other time. Maybe should try that. Will save lots of time haha 
I make chemically competent cells using rubidium chloride. I grow the cells for a few hours, then go through a couple washes and snap freeze them in liquid nitrogen. I store them at -80 for upwards of a year or more and see no real decrease in transfection effiency. I heat-shock transform all my ligation products. It is just so quick and easy.
If anyone is interested, I'll post my protocol.
Beverly
Here's an online link to the competent cell prep Liz mentioned.
Ultracompetent E. coli
This protocol is based on the Inoue protocol, as well, with slight modifications
I grew at 20C, inoculated with 1 ml of dense culture, and it took about 27 hours to reach the proper OD.