how can one get rid of genomic DNA in miniprep - (May/04/2006 )
hello, i did TA cloning, obtained nice number of white colonies. But when doing miniprep, i got three bands; which i think first one is of genomic DNA reason is that it appears above the plasmid size. Im doing manual prep using Alkali lysis method, and my insert size is 500bp. How can i get circular plasmid without genomic impurity so i can have good digestion?
main contamination issue, is when you add the lysis solution and agitate two vigourous. Inversing gentle 5-6 times the tubes does the jpb. If you think that it's not sufficent, let dissolve for 2-5' before adding the third solution.
I agree completely with Fred's diagnosis and recommendations. The most frequent cause of chromosomal DNA contamination in a miniprep is that you were too enthusiastic at the lysis step -- gentle works much better...
By the way, your miniprep is contaminated with chromosomal DNA, not genomic DNA...
I also agree with the other's recomendations for avoiding contamination, but I have an additional thought...are you running undigested plasmid? Three bands sounds like you might have nicked circular DNA (runs larger), linearized (runs at the expected size) and supercoiled (runs smaller) rather than contamination.
Fred and Homebrew are right about gentle lysis. But, it would be usefull indeed to check wether you just put your extracted plasmid on gel (because you would have to see three bands). If you cut your plasmid with an RE that cuts just once, you should see 1 band, if not you got contamination.
ushra, may you please attach a picture in order to determine if there is a possibility of undigested plasmid rather chromosomal contamination?