Recently I somehow overgrew the bacteria in LB and get a low yield when extracting DNA.
To get the highest yield, how many hours is should I grow bacteria?
And how can I know when the bacteria is best ready for mini/midi/maxi? Is there any way to measure the state of the bacteria? Thanks
In my experiment, it was 12 hrs to get more yield for E.coli. On the other hand, one of my labmates got good yield after 6 hrs. You could add some glucose like (50 gm glucose+ 50 mL DH2O mix) and add 1mL from this solution to 50 mL LB broth. Sometimes this method works very well.
Hi Wjkp,
-High copy number plasmids should only need to be grown for 12-16 hrs on average whereas low copy plasmids needs 18-30 hrs typically.
-Try growing the culture for 16 hours at 37 C, as some bacterial cultures may grow more slowly.
-Double check that the antibiotic in your LB media matches the antibiotic resistance on your plasmid.
-If the bacteria on your LB agar plates are not fresh, you should streak your bacteria onto a new LB agar plate before growing in liquid culture.
-More aeration may help to increase the density of the culture. Normally cultures shake at 150 - 250 rpm, increase this to 350 - 400 rpm to obtain a higher cell density.
-You can check bacterial concentration ready to use (mini/maxi/midi) by checking OD(optical density) using Nanodrop/spectrophotometer or following the protocol specified by plasmid extraction kits.
Hope this helps some of your concerns. Best wishes!
Consider factors like
1) High copy 8-16hr, low copy more than 16hr
2) Inoculum is inversely proportional to time
3) Antibiotic stability and concentration in case of Amp/ Zeocin etc.
4) OD before termination can be 1.5-2. more than 2 is not good
5) Use Swing bucket rotor for good yield
Consider all the factors mentioned by CHANDRA SHEKHAR DASARI and avoid using more than recommended volumes of cultures per column when you follow the column based plasmid isolation. It holds good even when you follow manual centrifugation based method.
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