Hi everyone I am using the OMEGA Endo-Free Plasmid DNA Maxi Kit to extract some plasmids for example pMD2.G, PCMV-VSV-G and pRSV-REV. These are high-copy number plasmid in bacteria and I followed instruction book step by step after growth and culture bacteria using 100ml LB liquid medium. The end products concentration is about 63.6ng/ul, 97.8ng/ul and the highest one is 208ng/ul.
So I am confused with the results.In my oponion, will Amp+ denaturation, as the antibiotic added in LB liquid medium, accounts for one reason for the experiment results?
Does anyone who have suffered this problem or know how to make improvement and solve it? Thank you.
When you elute the plasmid from the plasmid filter (before precipitation), put the same elute back to that filter and wait for it to pass through again. When I tried this, concentration was double as compared to the previous one.
Hi Monga, It's sounds good. I know that we can add more elution buffer to get more plasmid but the problem is the concentration will lower. This proctol seems like solve that problem. Really sincerely thanks for your help, I will have a try next time.
Hi Bao, I am not asking you to put more elution buffer. What I want to say is to use the same elution buffer which has been eluted already. Concentration would be almost double if you do that. Please do not put more elution buffer. Obviously it will decrease the concentration.
Let's say you eluted with 50 ul of elution buffer. Then you need to put back the same elution into the plasmid filter again. This process may take 10-15 mins more, but really effective.
HI Monga! This is a good idea, and I never thought about it. I should also try and see if it works. Thanks for clarification as I also did not understand it in your initial explanation.
It's works quite well. I have done an experiment to test it, the re-wash can increase about 30% to 40% concentration compare with primary concentration. By the way, ddH2O works as well as elution buffer. Really thank you Monga!
try PureLink Fast midi or maxi low endo kits. 0.4-1.5 mg yield in 30 min
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