I am trying to isolate plasmid from Agrobacterium rhizogenes LB510 and 072001. My promotor says to me that its plasmid is still wild type, not binnary or modified plasmid. I was searching for the bacterial strains but I still can't find these strains, so the plasmid (plasmid opine gene, plasmid size, and the sequence also) couldn't be found. What type of opine of wildtype plasmid is it?
Sorry in advance since I won't be able to answer your question, but I wanted to know what kit you were using in order to isolate plasmid DNA from Agrobacterium? I've been using the Qiagen miniprep kit but always get really low yields - hopefully you know of a kit which is optimized for Agro. Thanks.
I always successfully isolate plasmid from my Agrobacterium tumefaciens LBA 4404 using alkaline lysis-based method. It always work for me. I think wild-type Ri-plasmid is low copy number in nature, so it will produce low yield of DNA in normal miniprep plamid extraction. For your situation, I suggest you to use an extra amount of starting material, in E. coli 1,5 ml bacterial culture is enough for miniprep procedure, but for your Agrobacterium maybe you can use 3 to 5 x 1,5 ml culture (24-36 hours culture). Centrifuge the cell in separate 1,5 ml tubes then perform cell suspension, lysis and neutralization steps in each tube (as normal) and then perform ethanol precipitation, suspend the pellet DNA into small amount of water (10 ul per tube) then mix all of plasmid DNA from each tubes into one. Hope this can help you.
I dont have any idea about your Agro strain but you could ask your supervisor/promotor or person who become the source of your strain.
Best wishes.and good luck!
P.S.
This is the original paper of the alkaline lysis protocols:
http://nar.oxfordjournals.org/cgi/content/abstract/7/6/1513
Here some articles that may be helpful:
http://bitesizebio.com/articles/the-basics-how-alkaline-lysis-works/
http://bitesizebio.com/articles/plasmid-v-genomic-dna-extractionthe-difference/
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