5 replies to this topic

[Ecoli0157]

Hi,

I've just made DH5a competent cells for the first time. YEAH~~

The problem is that I was expecting library quality efficiency, but when I did a test transformation, colony numbers dont look that high.

This is what I did.

Into 100ul of cells, add 50 pg of control DNA ( I pre-chilled the tubes and incubation on ice and all other standard transformation steps)

Heat shock at 42oC 50sec

Recovery in SOC with incubation at 37oC 1hr

Plated 100ul of 1:10 and 1:100 dilutions on LB/amp

I got about 10 colonies and my calculation gave me about 2X10^7 CFU/Ug DNA( is it correct?)

Would you guys use these cells if you are in for g.DNA library? It is probally worth saving for PCR cloning and other general cloning experiment.  I think...

Thanks,,,

[phage434]

It's a little low.  I agree with your calculation.  What protocol did you use?   You should be able to hit 1e8 to 4e8 or so with a good protocol and care.  You may want to electroporate for a library, where it is hard to do worse than 1e9.

[memo]

If I use home-made competent cells I always use a transformation enhancer to be sure that I have enough cfu's. Greetz Me

[Ecoli0157]

Could you tell me what transformation enhancer is?

Also, I just noticed that the digital reading on my water bath was about 2.5oC lower than the thermometer.

Any idea how important it is to be EXACTLY at 42oC in terms of transformation efficiency?
(I would have been at around 44.5oC during the heat shock.

[HomeBrew]

The heat shock step is not critical. I've (inadvertently) skipped it many times -- you should still get transformants, though I don't know whether skipping it might have an impact on your efficiency (I don't worry about that because for most of my transformations, a single correct colony is all I need, and homemade competent cells have worked just fine for me in this regard).

I doubt that 50 secs at 44.5 C would have any measurable effect on the viability of your recipients.

[memo]

Ecoli0157, on Mar 11 2009, 03:56 PM, said:

Could you tell me what transformation enhancer is?

Also, I just noticed that the digital reading on my water bath was about 2.5oC lower than the thermometer.

Any idea how important it is to be EXACTLY at 42oC in terms of transformation efficiency?
(I would have been at around 44.5oC during the heat shock.

Hi I heat shock between 38-40°C and most of the times it works fine. I am using a solution with lipids as an enhancer named traen. Just google it and you will find the company which sells it.