about purification of large plasmids

methods at 163.NET methods at 163.NET
Thu Apr 23 11:55:44 EST 1998


Dear netters:

I have met a problem when trying to purify a large plasmid from low melting gel. The size of  this plasmid is about 40Kb. It seems every time I can only load a little amount , if the gel well is loaded too much, the plasmid seems can not  run out of the well. And even I cut the band to purify, the effeciency  is too low. Then I try the method of  sepharose-CL4B, this does not solve the problem, the plasmid is always mixed with genomic DNA and RNA.

So I wonder if anyone with the experienec on dealing with large plasmids can tell me:
1. In what way can I get purified plasmid, 
2. What elution buffer should be used when using sepharose to purify plasmid (I use TE with 0.1 M NaCl)
3. IF there is the possibility that what I thought was genomic DNA is actually the oligo-aggregation form of this large plasmid.

Any idea is welcomed and especially time- and money-saving ones. : < ) contact me at methods at 163.net

Ray
from methods at 163.net

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