Hello to everyone!
I have an insert that I transformed into E. coli STBL3 strain. These are liquid cultures that I stored at -80 ° C.These are liquid cultures that I store at -80C. I wonder, after the mini culture is completed, I get some of it.
Then, to start the maxi culture, I have to keep the liquid bacterial culture I received in the eppendorfa for 4-7 hours.
Is there any harm in keeping it in an eppendorfta + 4C for 4-7 hours in LB?
4-7 hours later I start the maxi culture with this.
What other way can I do this.
There is no problem in keeping it in 4 c for 24 hrs. actually its better than freezing and thawing. However, I recommend you to keep it in -20 C just in case you decided to delay your experiment.
I agree to the aforeentioned comment. 24 h at 4°C should not (or max. slightly) affect the outcome of your maxi prep.
This should work fine.
However, do streak out your culture onto LB plates to check purity. By the time you get to the maxiprep, it will be a third passage liquid culture, so there is a significant chance of contamination.
Hi Çiğdem ,
I agree to Elsayed, freeze thaw cycle can affect the outcome than keeping them at 4 degree and using them in few hours. You can keep them at 4 deg for several hours.
Çiğdem Yılmaz < Is there any harm in keeping it in an eppendorf at 4C for 4-7 hours in LB? 4-7 hours later I start the maxi culture with this.>
That is perfect ok. There is no reasons to place it in a freezer (-20 or -80C).
You can keep at 4oC with no effect on its viability as can resume growth well at 37oC.
Hi, dr
There is no harm in that if you want to keep the liquid bacterial culture for 4 to 7 hours in the refrigerator. You can make sure of its purity by making a subculture of it selective media to avoid microbial contamination
Çiğdem Yılmaz There is no harm to kept the culture in refrigerator for several hours. Usually I keep the pellet only and the concentration and purity turn out good.
Whatever transformation experiments you do always keep a stock of the bacterial culture in 15-20% glycerol and store at -80°C. If you need to harvest your plasmid constructs, just streak out a loopful of your glycerol stock on (antibiotic +) selection agar plate... Pick a single colony and grow your bacterial culture, harvest and extract/purify your plasmids from there. It is always a good practice to prepare plasmid stock from freshly grown bacterial culture.
But if you don't have the time to immediately do the plasmid prep, spin down the cells, remove the supernate and keep the pellets in the Nalgene tubes and store frozen at -20 or -80°C. Proceed with the DNA extraction/purification from there.
I missed the part that what you have a frozen bacterial culture, thawed then kept at 4°C while you proceeded to do a miniprep of it .... to test whether you have the right plasmid?
Normally we start by doing a small 3-5 ml bacterial culture, harvest the cells and do minipreps. Check whether you have the right plasmid then proceed to prepare bigger volumes.
So you basically have a lysed bacterial stock sitting at 4°C few hours before making maxiprep. Likely to have lesser yield for that.
i've done a lot of testing like this in early days some 30 years ago, and here is what i found: viability of a liquid culture (which you have not reduced to a pellet) in a flask or bacterial culture tube is better if you stick them on ice. Jut take an ice bucket or better, a styrofoam box with a lid, fill with fresh ice, stick your minipreps or maxiprep, and ice it for 24-48h, the cultures are great and viable. Just make sure the ice wont melt, or your flask will tumble and will spill everything into a container. To avoid this, kee the ice bucket in a cold room or in a fridge, covered with foil (which keeps the temperature very well. Or, cover with a styrofoam lid, and keep at 4C. I used this trick for years when i was multitasking and doing many things at once. Once you spin your bacterial cultures, you can freeze the pellet after LB. Just dont freeze the culture -bacterial walls will crack, releasing the plasmid (when you thaw). However, once you secure a pellet (after spinning your culture), freezing is actually good and helps to break the strong bacterial wall, people claim the yield is actually better. I never compared but i've frozen pellets many times and its a great way to halt an experiment, if you have other things going on. Last, if you want to chill the culture for few hours only, and then start maxi-culture, remember that your mini-culture wont be in this log phase recommended by Maniatis bible, it will take some time for bacterial to restart division. Hope it helps, good question
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