I am trying to grow an unstable plasmid (PERK, transmembrane protein of the endoplasmic reticulum) but the amount of DNA that I usually get from bacterial cultures is always too low (sometime enough only for an experiment).
I tried to grow bacteria (3, 5, 7 mL) at 30 degree overnight and overday monitoring the absorbance and no matter how many LB I use, I get always low amount of DNA.
Any suggestions?
Higher volume culture. Be careful too high of [antibiotic], especially kanamycin.
I usually only let the bacteria grow for 16-20 hours for optimal results, growing it any longer than that will be detrimental to whatever experiment it is you need to use the plasmid for, especially purification etc. For our plasmids, an antibitioc (Amp) concentration of 20 ug/ml is usually sufficient, although I culture in 100 ug/ml to ensure that the bacterial/plasmid cultures are pure. You shouldn't really use concentrations of antibiotics higher than 100 ug/ml. Also, you may want to use a larger flask to culture your plasmid (Rotary shaker, 37*C for most plasmids, 250-300 rpm), as this allows for better aeration & growth. Generally, I will use a flask about 10 times the volume of the culture. Have you tried using SOB/SOC/Terrific broth instead, to see if this helps? I would follow the Maniatis protocols in vol.1 of the CSH Molecular Cloning books, they always produce great results. I just used a bigger flask. Also, I don't usually spec my cultures, as I have done this in the past & the absorbance readings were too low when it was clear that the plasmid had grown because the culture was pretty cloudy (20 hrs). I didn't want to let it grow for too long, so I did a Plasmid Prep & yielded really pure DNA & attained the max. yield. So, maybe just keep your eye on it after the 16 hour mark, it should grow slowly, and then very quickly/become quite cloudy - this is the best time to harvest the bacteria. In saying all this, for a 3 ml culture... I would only grow that for 6 hours before inoculating 100ml overnight (16-20 hrS)...
I will suggest you to standardize the incubation period of the bacterial culture. Overnight incubation will serve sufficient, because as you proceed with again over day incubation cells may started to enter a late stationary phase or even decline phase. A 16-20hrs incubation was so far good for me when i was doing the plasmid isolation works. And one more thing, to get good quality of plasmid, you should always use a antibiotic stress. Under antibiotic stress say 50ug/ml Ampicilin you may get good results. Good luck.
Expression plasmids are usually low copy. Why are u usin 30oC? Make sure u use a proper bacterial strain for DNA purification. The strain u use for protein expression will not give you enough DNA.
Ok, thanks for your advice.
I was growing them under antibiotic selection, at 30 degrees overday+overnight, because it's a plasmid that is detrimentral for itselfs. So the Company (addgene) suggestes to grow it at a lower temperature for longer time. But definitely I should try to monitor better the time and maybe I should use SOB instead LB.
THANKS
Daniela,
You might try this. I did it before. I do culture the plasmid (100microliter) in 10ml LB for 16 hours or one day, on the second day take 2 ml out of the 10 ml and pipet them into 400 ml LB and leave them one day and on the second day do maxiprep instead of miniprep.!!!
the question of Christoph is super good, I had also experience using different bacterial strains and got different efficiencies.
sorry Daniela, you can also use midiprep which is in between the mini and maxipreps. best luck
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology