how to extract plasmids leaving bacteria cells intact - (Apr/20/2008 )
Dear All
I was wondering if it is possible to extract plasmids from bacteria cells without any harmful damage to the cellular system! could anyone share ideas if possible on how to go about!
thank you!
I was wondering if it is possible to extract plasmids from bacteria cells without any harmful damage to the cellular system! could anyone share ideas if possible on how to go about!
thank you!
I'm curious as to why you'd want to do this. At any rate, the problem is that plasmids are contained inside bacteria, and they aren't naturally excreted into the media. To purify plasmids, you need to release them from the cells by lysis. This is generally done by alkaline lysis or boiling. Afterwards you don't have any intact cells.
Ginger
Maybe this will work for you: if you figure out how to clone it into a phage vector and then extract plasmid in phage genome. Phage particles will be released into medium. no lysis step needed.
Hey Genehunter could you please explain me in more detail please? Actually i was so curious as this question has been raised in one of our conference here!!! thank you again and thank you inadvance for those who forward their ideas!
Eminov
Do you want to use the plasmid or generate plasmid-free cells?
What is the origin of replication and what is the antibiotic resistance?
What is the origin of replication and what is the antibiotic resistance?
Actully Tfitzwater its to generate a plasmid free cells.
Curing of a plasmid from E.coli using high-voltage electroporation.
D M Heery, R Powell, F Gannon, and L K Dunican
Nucleic Acids Res. 1989 December 11; 17(23): 10131.
is available free at http://www.pubmedcentral.nih.gov/pagerende...amp;pageindex=1
Acridine Orange has also been used http://www.pjbot.org/pjbot/samplecopy/pdfs/rasooletal.pdf
Otherwise, growing the cells in very rich media without antibiotics through several successive transfers will usually cure the cells. High copy number plasmids may exhibit reduced copy number at 20 to 30C, thus making this technique easier. After 2 or 3 successive overnights, streak out colonies on no antibiotic plates and grow overnight, then grid onto antibiotic-free and plus antibiotic plates to find the cured cells.
D M Heery, R Powell, F Gannon, and L K Dunican
Nucleic Acids Res. 1989 December 11; 17(23): 10131.
is available free at http://www.pubmedcentral.nih.gov/pagerende...amp;pageindex=1
Acridine Orange has also been used http://www.pjbot.org/pjbot/samplecopy/pdfs/rasooletal.pdf
Otherwise, growing the cells in very rich media without antibiotics through several successive transfers will usually cure the cells. High copy number plasmids may exhibit reduced copy number at 20 to 30C, thus making this technique easier. After 2 or 3 successive overnights, streak out colonies on no antibiotic plates and grow overnight, then grid onto antibiotic-free and plus antibiotic plates to find the cured cells.
Thank you so much guys it is been so helpful. Actually this whole idea is to curate indiginous plasmids and replace them with newly modified ones. Attempts will be done with either of the above three suggestions!