Why Ligation and transformation not working? - (Sep/28/2007 )

As I used to Ligation insert my DNA(PCR products) into pGEM.T.Easy, I transformed it into XL-2.
Finally I cloned it in agar plate overnight. So it suppose to grow White colonies in agar plate (Blue/White selection).
However, I got nothing in the plate today!! neither Blue or White colony.

So I been asking Why Why and WHY!!????

Here some of the reasons that I could think of because I made some changes in procedures.
1. Higher volume of PCR products in Ligation (3ul instead of 2ul)- I have around 50ng of DNA)
2. Higher of Ligation products used in Transformation. (10ul instead of 2ul)
3. More LIKELY!!- I incubated the mixture of ligation products, XL-2 and SOC medium for 30mins with 650rpm instead of 90mins with 150 rpm.

The reason why I used higher volumes of DNA was because of "More Than Enough"!
The reason why I used less time for transformation because of time limit ( I had to go soon).

What do you think the reason that I got no colony even in control would be???



Also I would like to ask how much IPTG(1M) you used to prepare an agar plate with 360ml of "LB medium with agar"?
I used 180ul but not sure if this is enough. (or 1.8ml??) Is this one of the major factor drave me to failure??

Thanks everyone!!!!!!I WOULD LIKE TO HEAR ANYTHING FROM YOU!!

Anyone has any idea??

Hello Nwt-X,

you already mentioned a few things...

1. Using 50ng in your ligation is okay (in a 20µl mix). It is recommended to use DNA in a final concentration of 1-10µg/ml. A too high concentration of DNA may result in only linear molecules.

2. You should use 1-5µl of your ligation for 50µl competent cells. Using more decreases the transformation efficiency.

3. The bacteria need some time... the shaking is only to support the cells equally with air and let them have consistent conditions. Increasing the shaking speed doesn't let them grow faster...They need at least 45min...

About the X-Gal and IPTG: In our lab we spread 40µl of X-Gal-Solution (20mg/ml DMF) together with 4µl IPTG (0,2mg/ml) on already poured plates.

Some questions to find other reasons for the failed transformation:
How did you do your ligation in detail? For example when your ligation buffer contains PEG you have to clean it up because PEG may inhibit the transformation. Additionaly PEG decreases ligation efficiency over time...

I hope, I gave you some clues...
Greetings,
Chakchel

Chakchel made some good suggestions.

Nothing growing on plates has happened to me twice before.

The first time, someone had put a much too high concentration of ampicillin when making the plates... This prevented ANYTHING from growing on them.

The second time, the bacteria itself was no good. The whole batch of E.coli that I ordered from invitrogen was dead. I found this out by using another batch of the same brand of E.coli that did grow.

It could be any little thing that's screwing it up.

Good luck!