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How do I make more competent cells? - by growing up and freezing glycerol stock? (Jun/26/2005 )

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Hi, I've been reading Promega's protocol for making more chemically competent cells and they say that for transformant buffer 2, you need 100mM (www.promega.com/guides/subcloning_guide/ _row/transforming_bacteria_row.pdf )

However, the protocols posted here and somewhere else say 10mM and I believe that this is the right amount. Could anyone confirm this for me? (I'm already doing my transformant buffer with 10mM MOPS)

-gsamsa-

Well, so far for heatshock transformation, I stick with the Calcium Chloride method! It's really simple and cheap!

so far, transformation efficiency is good! I have never used any media other than LB broth!

The key point should be noted that, if the cells was harvest at early phase (OD600<0.3) the competency is better than keeping the cells until higher OD!

-dtle-

Hello martina,

I was wondering if the only way to find that protocol is buying the book.
Thank you.


QUOTE (martina @ Jun 28 2005, 08:32 AM)
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.

-medchemgirl-

QUOTE (medchemgirl @ Jun 19 2006, 06:02 PM)
Hello martina,

I was wondering if the only way to find that protocol is buying the book.
Thank you.


QUOTE (martina @ Jun 28 2005, 08:32 AM)

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 used Ca-competents cells, they work fine:

1. Inoculate 100 ml SOB medium (LB-may be used) with a single colony or a 0.5 ml of culture, incubate at 37°C to OD600 0.3-0.4
2. Transfer the bacterial cells to sterile, disposable, ice-cold 50-ml polypropylene tubes. Cool the cultures to 0°C by storing the tubes on ice for 10 minutes.
3. Recover the cells by centrifugation at 2700g for 10 minutes at 4°C.
4. Resuspend pellet by swirling in 30 ml of ice-cold CaCl2 solution.
5. Recover the cells by centrifugation at 2700g for 10 minutes at 4°C.
6. Resuspend the pellet by swirling in 4 ml of ice-cold 0.1 M CaCl2

Now, I'm using high-competents cells (Hanahan Method). They works much more better!!

-aztecan princess-

Is those came from single colony if yes u proceed to inoculate and keep it for overnight/for some time(4-5hrs) and then inoculate in to fresh media this u can use it no probm
All the best

-Add colour 2 ur life-

The difference between electrocompatant and chemical compatant cells is the salt concentration. High salt concentrations cause improper electrical discharge on the electroporator (harming the divice and giving low transformation yields). Theoritically you can but depending on your cells and what they were designed to do they may be more adapted to the heatshock method.

-corkinsme09-

hi ,
a machine engaging in speciality fermenting bacterica largely can help you solve this problem ,the bacterium are enough for you to make more competent cells

-pandagxp-

I have ever used the online protocol of NEB website to make the competent cells and succeeded. but I don't recommend the protocol. I would like to recommend the protocol to make super-competent cells in the third edition of molecular cloning 3.


Do not grow the cells on Amp+ plates, you will get nothing.

-tulip1-

QUOTE (GPCR @ Jun 26 2005, 05:45 PM)
I have some BL21DE3 bacteria for expression. How do I make more of it? Can I just grow some overnight, dilute and let it grow until mid log phase and take some for glycerol stock? When these are thawed, will they be the same cells that can be used for transformation and expression?


... I think that if you are using a traditional mating method, you could potentially prepare more "competent" BL21DE3 juts by growing more of them smile.gif

QUOTE (fred_33 @ Jun 28 2005, 11:55 AM)
...
So i assume they've been made competent by the 10%glycerol wash? blink.gif

Fred


When you prepare cells for electro transformation, you do not render them competent, I guess you oly wash the salts away...and the electric current then renders the membrane porous, allowing DNA uptake.

-ph3no-

QUOTE (GPCR @ Jun 29 2005, 09:53 AM)
QUOTE (fred_33 @ Jun 28 2005, 05:55 PM)
hi
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? blink.gif

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?

-Kurtulus Buruk-

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