High molecular plasmid isolation form kit and alkaline lysis - (May/05/2012 )
Hi! I am having difficulities in isolating high molecular plasmid (>21 kb) using kit (geneaid mini kit). I'm use the same protocol but when I electrophoresis the plasmid, It looked like it was degraded in 23 kb area. But when I am use the alkaline lysis for isolate my plasmid, it have an intact band. There is another problem. My plasmid are 23 kb but when I electrophoresis it, it has two separate bands altough it may be a circular and linear plasmid but it has different size. One is in 23 kb but one is in 9 kb. Do anyone know this? Thank you very much..
Intact circular supercoiled plasmids will run faster than nicked or linearized plasmid, so you are probably seeing a mixture. You can test this by cutting your plasmid at a single location to produce only linear fragments.
How are you checking lengths -- at those sizes, gels typically have difficulty achieving high resolution, unless they are FIGE or PFGE gels.
Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
<*>Nicked open-circular DNA has one strand cut.
<*>Relaxed circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed (supercoils removed). This can be modeled by letting a twisted extension cord unwind and relax and then plugging it into itself.
<*>Linear DNA has free ends, either because both strands have been cut or because the DNA was linear in vivo. This can be modeled with an electrical extension cord that is not plugged into itself.
<*>Supercoiled (or covalently closed-circular) DNA is fully intact with both strands uncut, and with an integral twist, resulting in a compact form. This can be modeled by twisting an extension cord and then plugging it into itself.
<*>Supercoiled denatured DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.