I am doing plasmid isolation by Omega Plasmid DNA Minikit I #D6942-02 and Thermocycler GeneJET Plasmid Miniprep Kit #K0502. Tried both separately and no results.
I am following the instructions and using freshly prepared primary culture. All incubation periods and mixing either in the resuspension or in the lysis steps are followed carefully and I observe the viscosity of the solution after the lysis step. The white ppt. also observed after the neutralization solution. I do not know which step I might be doing wrong although my plasmid is around 6Kbp, so it does not require warming up my elution buffer. I am using sterilized distilled water as my elution. Cross checked the pipetting, chemicals, and the primary culture is really cloudy meaning that it is a rich bacterial culture. Any help?
Hello Fayrouz,
I don't know these kits precisely but in general, the elution buffer has to be slightly basic (like 5 mM Tris-HCl pH 8). Most of the time pure water is slightly acidic due to dissolution of CO2. Either equilibrate your water at pH8 with NaOH or use Tris as above.
Best,
Olivier
You might also want to remove a small aliquot (10-20ul) of the solution after the neutralization step and run on a gel to be sure there is plasmid present before you run your column. Be sure that your culture is actually E. coli and not some contaminant which wouldn't have plasmid in it.
Hi Fayrouz A Nossir ,
Ensure you use the selective antibiotic on your plasmid-containing bacterial culture. Use freshly prepared antibiotic solutions to confirm if your cells contain plasmid by culturing these on antibiotic plates, and select antibiotic-resistant cells and process them for plasmid isolation.
You may also try eluting it in TE buffer, but it might not really affect that much, NFW works equally good, unless you plan to store the plasmid long term at -80*C, where TE buffer might be better.
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