Yes, you need to both purify and linearize your plasmid DNA before you do your dilutions to create your standards. You use a plasmid purification kit, calculate the copy number, then account for that concentration when you set up your restriction digest. you do not use a PCR cleanup kit.

Hi Lena

In our lab we do not linearise the plasmid. I do not think this would be necessary for titrations. Also, the quality of DNA derived from PCR cleanup is often inferior to maxiprepped DNA, so I wouldn’t advise that either.

For buffering, I know that some groups dilute their plasmid standards in genomic DNA, but our lab found that this does not drastically alter the outcome (compared to plasmid buffered in TE).

So, a maxiprepped, circular plasmid, diluted in TE will be absolutely fine.

Hi Lena,

If you want estimate the copy number of lenti in transduced cells the ANCHOR technology fit prefectly. With this techno, you can very easly and precisely estimate the number of copy by cells, and the data are beautiful!

To resume, ANCHOR system allow to generate fluorecent spots corresponding to gDNA integrations. The number of spot and their intensity allow to calculate very precisely the number of copy by cell.

You can have a look here to know more about ANCHOR:

https://www.linkedin.com/pulse/first-ever-live-tracking-hiv-1-based-lentiviral-human-grandchamp/

Let me know if you want further information

regards

Nicolas