Hello

We have a problem in precipitating plasmid DNA (Exactly PIRES2-EGFP) with the PEG-MgCl2 method.

The mentioned plasmid after culturing in E.Coli bacteria, harvested and extracted via using of Alkaline Lysis method. Then, the extracted product (pDNA) prepared for further Purifications performing the PEG-MgCl2 method. The first steps of purification well performed and our plasmid had precipitated; but After adding PEG-MgCl2 to the pDNA, the product disappeared! even the centrifugation in high rpm status didn't work.

so the question is:

At this step, what we should do to reprecipitate the pDNA? PEG-MgCl2 optimization? How?

• PEG-MgCl2 Concentration: 40% (w/v) of PEG in 30mM MgCl2 ioniq solution)
• Centrifugation condition: 30 min at R.T (14000 rpm=11000 rcf)