Arabidopsis plasmid rescue

Hello,

I activation tagged the arabidopsis plant (Col-0) by vector pSKI015. First I used TAIL-PCR with all possible primers AD and 3 nested TDNA left border primers as described by Liu et at. 1995. But I failed many times in TAIL PCR with some interesting mutants . After then I tried for plasmid rescue with EcoRI for RB and Bam HI, SpeI for LB. In plasmid rescue, I got some (10-20) colonies per plate. This is more less numbers as than described by other authors. However, further I tested for reference by striking on LB plates containing 100mg/L amp plates, colonies were grew well and then subcultured it. The cell growth in amp containing media is excellent like pSKI015 vector. Then, I isolated Plasmid DNA by using alkaline lysis method and QIAGEN plasmid kit as well. But the Plasmid DNA precipitation is very low (it is impossible for digestion and sequencing). I also did midi prep the result is same.

Do you have any ideas, How to isolate enough pDNA from the plasmid rescued Ecoli cells.

Thank you in advance for your kind suggestion

Sincerely yours

Giri Tripathi
Korea





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