i have done plasmid extraction of bacteria with alkaline lysis methode. I'm using TOP 10 E.coli and pYES 2/CT as plasmid vector. but, when i run electrophoresis in agarose 0,8% 100V for 27 minutes, there is no band appear. i have repeated extraction 3 times but there is no difference. still no band appear. any suggestion?
addition: i culture the bacteria on 3 ml LB with 75 microgram/microlitter amphiciline and incubate them 37 celcius for 17--20 hours. the medium turn very turbid.
It seems to me you have a contamination, re extraction by using different conditions
regards
Please give more details like a pic from your gel
Also consider that your time of running was not enough.
So your gel and run is good.
I strongly suggest you increase time of running because you are running a high MW DNA.
Also protocol for running total DNA says at least 40 min you need
I can recommend you two things:
1. Instead of a 0.8% gel, make a 1% gel and load 12µl of your samples.
2. Make a 2.5% thick gel and load about 20µl of your samples.
this can help you to get better bands.
I would recommend using a known control strain from your lab stock or from another lab and grow your strain and the control strain side by side and extract plasmid. That way you can determine whether the problem is with your strain, or with your extraction method.
pYES2/CT is high-copy-number in E. coli. It may happen it produces enough beta lactamase to destroy all ampicillin in culture medium within first several hours. Then, the plasmid might be lost because it's not needed any longer for viability of the transformed strain while its maintenance cost is high. I would suggest adding fresh ampicillin to the final concentration of 100 mcg/ml 3-4 hours after inoculation (and possibly later too).
Victor G Stepanov thanks in advance, i've expected it before. i thought the medium turn very turbid and the bacteria destroy all the amphiciline and they throw away the plasmid because they're not needed any longer
While Victor is correct in that the ampicillin is degraded rapidly during the growth of a culture, that is always the case nonetheless people isolate amp-R plasmids all the time. So unless your plasmid is especially toxic to the cells, I don't think that is the problem.
May be DNA concentration is very less you can take more amount of DNA . or modify the protocol what you are following now.
All the best...
It might be your sodium hydroxide solution. NaOH absorbs CO2 from the atmosphere and this seems to interfere with plasmid extraction. Making NaOH fresh from pellets often seems to help.
dear all, the problem has been fixed. my colony isn't e. coli anymore but it turn out to be another species
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