purposes of uncut plasmid DNA - (Jul/28/2007 )
hi!! i did gel electrophoresis after plasmid extraction last wk,a senior student explained to us the importance of uncut plasmid DNA....but i still dont comprehend the purposes of these supercoiled,nicked and linear plasmid dna....quite confusing.....would be very glad if anyone could help me solve my problems.... i`m a biotech sophomore,but still very stubborn to making discussion of my experiment results
I am as puzzled as yourself. For most cloning purposes plasmid DNA in its uncut form is not very informative. YOu can't even tell the plasmid's size. Linear DNA (in the ladder) runs differently from supercoiled DNA (which the plasmid will in in)
Perhaps your senior was explaining the various forms of plasmid , in the context of quality control. If that is so, think about it this way. Only fully intact plasmid, as in supercoiled plasmid is any use to you. Damaged and broken plasmid DNA is of little use, they don't replicate accurately, mostly useless for PCR and unusable for further ligation work. So by running raw plasmid out, you can tell if your DNA preparation has been conducted successfully.
Now on the other hand, nearly all modern cloning e coli strains, have been engineered so that production of native endonucleases, and recombinases have been mostly eliminated. Thus it is no longer so important or difficult to conduct a good DNA prep.
tat makes sense, i`ll take the part tat says abt `damaged plasmid dna and replication problems` thanks 4 the reply
My old PI used to suggest running undigested DNA after cloning to have an idea if the ligation worked or not. But the insert has to be more than 10% the size of the original plasmid, i think. This way, you can save some enzymes and donot have to do a lot of miniprep digests. But can be time consuming.
hi,there... hey,u r right!! today my senior asked me to further analyze the plasmid DNA i had extracted using restriction enzy[Hind III & EcoRI] and also uncut plasmid without restriction enzy ....the expected result is tat if i hav got successful insertion of the plasmid, there wld b 2 bands on the gel.now i know the importance of uncut DNA....