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Poor Plasmid Prep Yield

Poor Plasmid Prep Yield - Molecular Cloning Forum

Poor Plasmid Prep Yield - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 02-10-2009, 07:45 PM
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Unhappy Poor Plasmid Prep Yield



I知 trying to do a cheap, in house plasmid prep (i.e. no kits) by standard alkaline lysis of a pCMV plasmid.

I知 currently using Qiagen P1, P2, P3 formation buffers for lysis. Precipitation in IPA. Suspension of DNA in TE, RNase digest, Phenol Cholorform extraction, re-precipitation in IPA.
No matter what I try I get really (REALLY!!) high A260 and A280 values but very, very low yield by AGE with heavy high molecular weight contamination with no noted RNA on the gel.
I assumed that it was chromosomal DNA contamination though I知 not sure how/why it would happening, my lysis step seems normal, avoiding precipitate, etc.

Not sure what is going on here. Previous attempts to prep this plasmid by ENZA Omega BioTek kit didn稚 go well either. Pour yield but it was clean.

Should I try PEG to clean up my prep? Or does this just help with RNA contamination?
Any suggestions would great!
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