Disappearing plasmid - (May/19/2010 )
I'm working with soil isolates which might be pseudomonads. These strains exhibit copper resistance as well as antibiotic resistance so i proceeded to plasmid extraction. My gel looked something like this; a feathery chromosomal dna band followed by two sharp bands and then a long smear (presumably rna and fragments of chromosomal dna). I consistently obtained the same results for about 3-4 times when i repeated the extraction but after that the plasmid bands (incidentally the chromosomal dna band as well) just disappeared. I've been trying for over a month now but without any avail. I had used alkaline lysis procedure for the extraction. the strains grow really well in media supplemented with ampicillin or copper or both but do not give any plasmid recovery.
i would really appreciate if anybody could help me recover those bands. im desperate.
p.s. i cut the preps containing plasmids with EcoRI and pstI. both of them degraded the feathery chromosomal dna band but did not produce any cuts in the two sharp bands (plasmids)
This is a really obvious suggestion, but you haven't included any info about it, so please don't get me wrong:
Are you using a selective agent in your media when you grow your bacteria?
Yes..i am. I add ampicillin to LB medium. I use 2 ml and grow the culture overnight (16-18hrs.) at 120-150 rpm. I set the shaker for 30C but since its really hot here, the temprature cant be maintained. so i guess its around 30 + 3C.
Are you doing plasmid preps, or genomic DNA isolation? If you're doing plasmid preps, you shouldn't have ever seen any "feathery chromosomal dna bands"...