help with making ultra competent cell (Inoue method) - (Jul/05/2008 )

Hello everyone,

I am trying to make ultra competent cell
According to Inoue's method (1990), 10^9 cfu/ug is obtained.
Transformation buffer (TB):
10 mM Hepes pH 6.3
15 mM CaCl2
55 mM MnCl2
250 mM KCl

I was trying the method many times but could only get 10^6, very bad.. has anyone tried the method and got really high efficiency?
A few questions:

1. Why is the TB needed to be filtered. Can you not autoclave it?
2. Why the salt has to be added as powder? Can you make a concentrated stock and then dilute when you make the TB? For example, make 1 M Hepes, 1 M CaCl2, 3 M KCl, 1 M MnCl2 then mix together?
3. Why have to adjust the pH first before adding the MnCl2?

sorry, it seemed that i have so many questions, a few more:
4. When resuspend the cell in TB, I got a big clump and it is really hard to resuspend, I heard that if you pippet the cell, it's bad, so how can you resuspend the cell while it forms clumps? Can you just add a ml of water, resuspend then add the TB?
5. When freezing in liquid N2, just drop the bottle of the tube into the liquid or drop the entire eppendorf into the liquid N2?

Thanks a lot everyone[video][/video]

I've never gotten the Innoe method to work well. After trying all of the techniques, this is the one which works for me: http://openwetware.org/wiki/TOP10_chemically_competent_cells
It also works for Invitrogen. Do what they do, not what they say.

The pH has to be adjusted prior to MnCl2 addition because adjusting the pH up (not down) yields a brown precipitate of manganese oxide (hydroxide?). This does not seem to change the behavior of the solution, however.

Use a flat bottom centrifuge tube to make resuspension of the pellet much easier. Resuspend *before* adding a large volume of buffer, when the tube is almost empty.

I've never gotten the Innoe method to work well. After trying all of the techniques, this is the one which works for me: http://openwetware.org/wiki/TOP10_chemically_competent_cells
It also works for Invitrogen. Do what they do, not what they say.

The pH has to be adjusted prior to MnCl2 addition because adjusting the pH up (not down) yields a brown precipitate of manganese oxide (hydroxide?). This does not seem to change the behavior of the solution, however.

Use a flat bottom centrifuge tube to make resuspension of the pellet much easier. Resuspend *before* adding a large volume of buffer, when the tube is almost empty.

I always use stock solutions, except for the calcium chloride, which is used at 80 mM, so that it is convenient to add as a solid.

Thanks a lot phage434... I will give it a try again