8 replies to this topic

[Kali_Kali]

Hi,

Please help me.

I performed E. coli transformation by heat shock.

But I have no colony.

My condition is as follows.

  - E. coli cp cell, 200 ul

  - 18E7 plasmid(1.4 ug/ml), 10 ul

  - Amp+(50 ug/ml) LB plate

 

The portions of plasmid is too small?

Or I have another problems?

 

Regards,

Kali

 

[phage434]

That should be plenty of plasmid DNA. Very likely the problem is the competence of your cells. Are they commercial or home-made? If made, how?

[Kali_Kali]

Hello.

First, Thank you for your comment.

The competence cells used to traonsformation are home-made by calcium chloried methods.

If competence cells are problem, I select another protocols?

First of all, I think competence cells culture once more.

[phage434]

Home made CaCl2 cells rarely perform very well. There are a variety of much better protocols, and some commercial treatments that will give dramatically better results. I'd recommend that you look at the Zymo kits for TSB competent cell production, or if you want to do it yourself, my favorite protocol is here:

http://openwetware.o...competent_cells

 

Easier protocols with acceptable results are here:

http://openwetware.o...competent_cells

This is similar to the Zymo kit.

[ibmbio]

As mentioned by phage434, the commercial are of high efficiency usually.

 

The recommended plasmid concentration you need: 1ul of 100ng/ul

The commercial CC DH5-alpha: 40-50ul

The ampicillin (or carbenicillin) concentration: 50-100ug/ml

[labtastic]

Home made CaCl2 cells rarely perform very well. There are a variety of much better protocols, and some commercial treatments that will give dramatically better results. I'd recommend that you look at the Zymo kits for TSB competent cell production, or if you want to do it yourself, my favorite protocol is here:

http://openwetware.o...competent_cells

 

Easier protocols with acceptable results are here:

http://openwetware.o...competent_cells

This is similar to the Zymo kit.

 

How would you compare the first protocol you linked for making competent cells to the Inoue method?

 

I've been doing the latter for years now and have been quite pleased with it. My basic litmus test is that my cells must be competent enough to do double transformations reliably, which these cells always are. But I am always looking for ways to improve.

 

I've got some experiments coming up where I need to do triple transformations, and certainly my Inoue cells won't be able to do that. I'm not even sure electroporation can do that...maybe? Luckily one of the plasmids will be consistent throughout all the cultures so worst case scenario I plan to make competent cells with the one plasmid already in it, then do double transformations into those cells. 

[phage434]

In my hands the CCMB protocol is more reliable and higher efficiency. I don't ever try to transform three plasmids at the same time, so I can't tell you about your likely success. I would probably choose electroporation for challenging coli transformation.

[labtastic]

Thanks.

[labtastic]

In my hands the CCMB protocol is more reliable and higher efficiency. I don't ever try to transform three plasmids at the same time, so I can't tell you about your likely success. I would probably choose electroporation for challenging coli transformation.

 

For what it's worth, the other day I tried transforming three plasmids at once with electroporation and it works great. Lots of colonies.