Stock not growing. Can i re-grow Plasmid from sample? - (Jul/04/2010 )

I recieved a gfp-tagged construct which i transformed into electrocompetent e.coli cells.
I then plated the cells on agar plates with kanamycin, to get growth of my specific construct.
I grew up one of the colonies in lb broth, and i did a plasmid purification step.
I also made glycerol stocks at the time, which i froze at -80C

That was 3 months ago.

I still have some of my purified plasmid left over, but i wanted to make more.
So i took some of the glycerol stock out of the -80C.
I took 5ul and placed it in a bacterial tube, along with 5ul of kanamycin and 5mls of LB broth.
After rotating for 8 hours, nothing had grown. After 24 hours, there was still nothing.

I tried a different approach and did streak plates of my stock.
Nothing grew after leaving it overnight. After 48 hours, i still saw nothing.


My questions are:

1) IS there something else i should try with the glycerol stock?
2) Is it possible to take the purified plasmid that i have, and repeat everything again, only this time, transforming the plasmid into ecoli cells instead of the construct? Would i go about doing this in the same way?

It sounds as if your glycerol stock is dead. For important strains, it helps to test the glycerol after -80 freezing. At this point, you probably won't get viable cells from it. Given that you have plasmid DNA it should be very easy to re-transform. For future reference, you should not throw out glycerol stocks that are dead, if they are valuable. You can run a miniprep on them to recover plasmid DNA as a last resort.

A typical way to fail with making glycerols is to omit vortexing the tube after adding glycerol and cells. There may be a spot in your tube that has viable cells, if this has happened.

I totally agree with phage434,

However, I share with you my last gamble choice: use all of the total glycerol stock, add into 6-8x volume of growing broth (and grow under shaking condition)...if you believe there might be still viable cells there. Try plate the whole thing after 48 hours. I know this may not be a good method, and although I had use this method for a few times to get some of my rare archive strains back, but not all the time I succeed in revive it. Still, this is something I tried before with some but not all success.

I have two questions so i can try to figure out your problem:

1) how did you make your glycerol stock? (how much culture vs how much glycerol)

2) when you made the glycerol stock and prepped your plasmid before, the prepped plasmid was the correct plasmid right? (you got your plasmid when you prepped the culture)

The reason i asks these are because for (1), if your glycerol stock is frozen solid in the -80C and you thawed it to take out 5ul, you may have killed your cells by thawing them. if your glycerol stock is frozen solid, the correct way is to scrape off some of the cells.

The reason i ask about question (2) is because you didn't mention a growth phase in liquid media w/o selection after transformation of your plasmid before you plated so i'm not sure if you did this. For kanamycin this is necessary or efficiency goes way way down so your colonies on your plate could be mutants that actually do not contain your plasmid.

molstudent on Jul 4 2010, 07:18 PM said:

1) I went with 50ul of the cells and 50ul of Glycerol. Would this have been too much, or not enough?
2) I'm pretty sure that the plasmid i ended up getting was the correct one.

3) I didnt let the stock thaw out fully, but at the same time i did let it thaw a little instead of scraping off the cells.

4)Sorry, i forgot to add that i let the transformed cells grow in SOC broth. This is what i plated out first. I grew colonies from the plates, and took a colony and grew it up in the LB broth.



I'm assuming now that my cell stock mightnt be too viable and might start again.
(I have very little construct left.
My protocol for transformation was to take 50ug of the construct and add it to 50ul of electrocompetent ecoli cells.
I mixed gently and incubated on ice for 30 minutes. I heat shocked the cells for 30 seconds at 42C and then put them on ice for 2 mins.
That was when i added 500ul of SOC.I put the tubes on a shaker for 30 minutes at 37C, then plated them.)


Does anyone have a protocol for transforming a plasmid into ecoli cells, or would it be the same as how i did it for the construct?


I might try a last ditch attempt to throw all the glycerol stocks into LB broth and keep them shaking at 37C for 48 hours to see if i can get anything again.

Thanks for the help so far everyone.

50ul cell and 50ul og glycerol seems very little but the ratio is fine. i usually do 500ul of each. for this ratio you should have scrapped the cells off instead of thawing it, thawing it may have killed your cells.

your transformation protocol seems fine but i incubate in SOC for 1 hour. heres how mine usually goes:

-add some plasmid to competent cells
-leave on ice for 5 minutes
-heat shock for 90secs
-ice for 2 min
-add 250ul SOC and shake at 37C for 1 hour

molstudent on Jul 5 2010, 06:18 AM said:

How much plasmid should i use? should i go with about 50ug?
Also, would 250ul SOC be enough, considering before i would use 500ul?