I extract Plasmid DNA by using QIAGEN Mini Kit. I was hoping to get one clear band at 5 Kb ( My Plasmid Size is 5kb). I used overnight grow culture and then isolate plasmid using QIAGEN Kit. Here I attached image which is showing 4 bands. In next step I want to do RE digestion but without getting pure single band, how could I go further?
In DNA ladder dark band is 5Kb.
Extra bands can occur when plasmid DNA is nicked , linearised and/or permanently denatured. Plasmid DNA that has been nicked (covalently opened) will run slower than supercoiled DNA during electrophoresis. A small amount of this species of DNA is common and is suitable for downstream applications. Permanently denatured DNA will migrate ahead of the supercoiled DNA and may not be suitable for downstream applications.
The most important measure: Do not allow the lysis reaction (Buffer P2) to proceed longer than 5 minutes. If possible reduce the time to 4 - 4:30 minutes.
Check buffers for nuclease contamination & replace if necessary. Use new glass & plasticwares, and wear gloves.
Plasmid DNA will be nicked/sheared/ degraded if DNA was poorly buffered. Redissolve DNA in TE buffer,pH 8.0, to inhibit nuclease activity and maintain stable
pH during storage.
To avoid shearing during redissolving, redissolve DNA gently, without vortexing or vigorous pipetting. Avoid using small pipet tips.
Hope these few tips will help to improve the quality of your plasmid.Good luck.
Extra bands can occur when plasmid DNA is nicked , linearised and/or permanently denatured. Plasmid DNA that has been nicked (covalently opened) will run slower than supercoiled DNA during electrophoresis. A small amount of this species of DNA is common and is suitable for downstream applications. Permanently denatured DNA will migrate ahead of the supercoiled DNA and may not be suitable for downstream applications.
The most important measure: Do not allow the lysis reaction (Buffer P2) to proceed longer than 5 minutes. If possible reduce the time to 4 - 4:30 minutes.
Check buffers for nuclease contamination & replace if necessary. Use new glass & plasticwares, and wear gloves.
Plasmid DNA will be nicked/sheared/ degraded if DNA was poorly buffered. Redissolve DNA in TE buffer,pH 8.0, to inhibit nuclease activity and maintain stable
pH during storage.
To avoid shearing during redissolving, redissolve DNA gently, without vortexing or vigorous pipetting. Avoid using small pipet tips.
Hope these few tips will help to improve the quality of your plasmid.Good luck.
Generally when you isolate plasmid you will get three forms of DNA i.e. nicked, linear and supercoiled. supercoiled DNa runs faster and linear will migrate at the expected size and nicked runs above linear. But here you are getting more than that, one possibility is that it s a contaminated with other plasmid. to test this you can linearize your plasmid preparation by cutting with single enzyme and run on gel if you get single expected band, then it is not contaminated otherwise you will get two different bands. Hope this will help you.
Krishna leela. I got 3 bands on gel electrophoresis. It means my extarction correct?
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