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Hello everyone,
I am currently doing a project that requires me to clone and purify BACs (~200kb). I can transform the BACs into the bacteria just fine, but I am having real trouble purifying the BACs so I can sequence. I have tried Qiagen's midi prep kit, and I tried a protocol that uses no column with a solution called STET, followed by a phenol/chloroform/isoamyl alcohol purification. Nothing seems to work - either the concentration is too low to sequence, or the sequencing reaction won't work at all (for reasons unknown to me). Has anyone worked with BACs before, and if so do you have a method to isolate them or any suggestions in general?
thanks
I am currently doing a project that requires me to clone and purify BACs (~200kb). I can transform the BACs into the bacteria just fine, but I am having real trouble purifying the BACs so I can sequence. I have tried Qiagen's midi prep kit, and I tried a protocol that uses no column with a solution called STET, followed by a phenol/chloroform/isoamyl alcohol purification. Nothing seems to work - either the concentration is too low to sequence, or the sequencing reaction won't work at all (for reasons unknown to me). Has anyone worked with BACs before, and if so do you have a method to isolate them or any suggestions in general?
thanks
