How to save my plasmid - I run out of my plasmid and my EColi doesn't grow (Apr/14/2007 )

Hi. I sequence my plasmid recently getting at last the right sequence in my insert. Unfortunately, I run out of my plasmid during the sequencing reactions, but I wasn't worry because I had frozen 1ml of LB transformed EColi with this plasmid (0.5ml of a 20ml miniprep). The problem is that I forgot to add Glycerol and I think my beloved bacteria are dead because I can't make them grow even without antibiotic.

To overcome this problem I think I could purify my plasmid from that 1ml, but I'm worried that maybe during the extraction process I can lose all the plasmid and I would have to start all over again. Other idea is taking just 100ul, incubate for 10min at 100C to release the plasmid and then use the supernatant to do transformation with new EColi because you only new a tiny amount of your plasmid for the transformation, isn't it?

What should I do? Do you have more suggestions? What could it go wrong if I do one or the other way? It is unlikely that mutations happend during this preparations, am I right? Should I sequence once more after doing all of this again? (Im so tired of doing sequencing...)

Thanks a lot.

Do you still have the vial that contained your plasmid?

If you still have the vial, there should still be residual DNA in it. (If you use eletroporation, all you need is 10pg or so of DNA.) Add your competent cells into said vial to wash off the DNA and transform the mixture.

That should give you colonies, a number of colonies



As for your plan, I assume you are going to do more then just cook 100ul of LB culture.
You need to remove the LB medium first, else it will interfere with the transformation protocol. I suggest pelleting the cells down and removing the spent media first.

And if you are working on a small stable plamid (anything below 10kb), you could try plating 100ul of your media on a fresh LB plate (+ appropriate selection of course). All you need is for a single cell to survive, and if the culture was saturated, well... even a survival rate of 1 in a billion, would still result in a few cells growing and forming colonies.


But the moral of the story... never get into a situation when you have run out of plasmid.

Thanks for all suggestions and advice.

Unfortunately, I threw away my vial. However, I will remeber for the future.

About the second suggestion of platting 100ul, I had tried 100ul in LB culture and bacteria didn't grow, so do you think that it is possible they grow in a plate. Is it not more or less the same, LB medium or a plate?

Cheers

well a plate is more sensitive. A single bacterium will produce a colony on a LB plate after an overnight incubation, but the same single bacterium may not cause the LB medium to become cloudy until after 2 days of incubation. An overnight incubation may leave the culture 'clear'.

However as you have already tried growing growing said bacteria in a culture, the odds aren't good. I assume you left the culture growing for nearly 24hrs, with no visiable change.

I guess you will have to try extracting the plasmid again.