6 replies to this topic

[spellberg56]

Hello, I am interested in making my own chemically competent cells instead of pissing away money on the pre made ones. The protocols all seem straight forward but they do not address the type of cells to use. Obviously e. coli, but what strain would one chose to use? Do the cells have to be free of plasmid DNA? In other words, could I use cells that I had previously transformed? Maybe the existing plasmid would be lost during the competency protocol. I use store bought XL1 blues normally? Is XL1 blue the strain name or what you call them after they have been made competent?


Thank you for your time.

Mike

[fishdoc]

spellberg56, on Nov 23 2009, 03:57 PM, said:

Hello, I am interested in making my own chemically competent cells instead of pissing away money on the pre made ones. The protocols all seem straight forward but they do not address the type of cells to use. Obviously e. coli, but what strain would one chose to use? Do the cells have to be free of plasmid DNA? In other words, could I use cells that I had previously transformed? Maybe the existing plasmid would be lost during the competency protocol. I use store bought XL1 blues normally? Is XL1 blue the strain name or what you call them after they have been made competent?


Thank you for your time.

Mike

If you have some competent XL1 blues in the freezer, they are your source. Grow some up, make them competent, freeze, repeat when needed. I think our lab bought some XL1 Blue MRF' cells in the late 90s. We use them frequently, but only bought them that one time. All you need is some LB to grow the cells in and a protocol to make them competent. Keep a tube in the freezer to inoculate from.

[spellberg56]

fishdoc, on Nov 23 2009, 05:20 PM, said:

spellberg56, on Nov 23 2009, 03:57 PM, said:

Hello, I am interested in making my own chemically competent cells instead of pissing away money on the pre made ones. The protocols all seem straight forward but they do not address the type of cells to use. Obviously e. coli, but what strain would one chose to use? Do the cells have to be free of plasmid DNA? In other words, could I use cells that I had previously transformed? Maybe the existing plasmid would be lost during the competency protocol. I use store bought XL1 blues normally? Is XL1 blue the strain name or what you call them after they have been made competent?


Thank you for your time.

Mike

If you have some competent XL1 blues in the freezer, they are your source. Grow some up, make them competent, freeze, repeat when needed. I think our lab bought some XL1 Blue MRF' cells in the late 90s. We use them frequently, but only bought them that one time. All you need is some LB to grow the cells in and a protocol to make them competent. Keep a tube in the freezer to inoculate from.

So am I to assume that by repeating the chemical competency protocol, that I am "refreshing" their transformation efficiency?

[swanny]

I wouldn't say you're "refreshing" the competency. You are making a fresh culture of cells with strong cell walls, then treating them to make the walls suitable for transformation expts.

[fishdoc]

spellberg56, on Nov 23 2009, 04:28 PM, said:

fishdoc, on Nov 23 2009, 05:20 PM, said:

spellberg56, on Nov 23 2009, 03:57 PM, said:

Hello, I am interested in making my own chemically competent cells instead of pissing away money on the pre made ones. The protocols all seem straight forward but they do not address the type of cells to use. Obviously e. coli, but what strain would one chose to use? Do the cells have to be free of plasmid DNA? In other words, could I use cells that I had previously transformed? Maybe the existing plasmid would be lost during the competency protocol. I use store bought XL1 blues normally? Is XL1 blue the strain name or what you call them after they have been made competent?


Thank you for your time.

Mike

If you have some competent XL1 blues in the freezer, they are your source. Grow some up, make them competent, freeze, repeat when needed. I think our lab bought some XL1 Blue MRF' cells in the late 90s. We use them frequently, but only bought them that one time. All you need is some LB to grow the cells in and a protocol to make them competent. Keep a tube in the freezer to inoculate from.

So am I to assume that by repeating the chemical competency protocol, that I am "refreshing" their transformation efficiency?

No, you're not repeating the competency protocol on competent cells. Competent cells are fundamentally E. coli cells. You culture them just like any other bacterium. Competent cells just get treated so that they will facilitate taking up external DNA. If you have a tube of competent XL1 Blue in the freezer, use it to inoculate 5 mls of LB (or whatever rich broth you have for culturing E. coli) and grow it overnight (12-16 hrs). In the morning, add glycerol to a final concentration of 15-20% and store the culture at -80 C. These cells are no longer competent, but they are a source for you to grow subcultures that you can make competent. So, what you'd do is use the freezer stock to inoculate a broth and grow overnight. The next day, use that culture to inoculate a larger volume of broth and grow to an OD of about 1.0. Then make that batch of cells chemically competent using a protocol from somewhere. I don't have a protocol for that as we do all our transformations by electroporation. Either way, there's no need to buy competent cells every time you need them. All you've got to do is grow some up and make them competent using a standard protocol.

[aimikins]

I would add a couple things here: if you've transformed the cells in a culture, don't ever re-transform them.  don't assume the plasmid is lost over time (that only happens when you DON'T want to lose it     )

also, which strain you choose depends on what you're doing downstream of the transformation.  look at methylation patterns or ability to blue/white screen for convenience, and consider whether you're using the plasmid as an expression vector.

[molgen]

aimikins is correct in all he/she wrote.

I want to clarify one thing,

Quote

which strain you choose depends on what you're doing downstream of the transformation.

That means that you pick your plasmid by what you want to do downstream (i.e. various tags, expression....). The strain you choose has to fit the signals that are on the plasmid.

Not all plasmids will grow in every strain.

Edited by molgen, 25 November 2009 - 02:37 AM.