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

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)

---

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!

---

Hello martina,

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

---

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!!

---

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

---

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.

---

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

---

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.

---

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



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.

---

---