Unexpected band in BL21 (DE3) plasmid isolation (View forum version)



james21

Posted 11 July 2016 - 09:27 PM

Hello!

 

I am working with a line of BL21(DE3) cells that contain a pET14b vector w/1.2 kb insert, resulting in the top band you see that is ~6kb.  The band directly below it is the pLysS plasmid (~5kb) that is in the DE3 line.  I had to run the gel forever to separate them as I only want pEt14b.

 

What might that third band be?  It is ~3kb according to the 1kB DNA ladder I am using.

 

Really curious as to what others may think, Thanks for reading!

 

James

  • 10 July 2016.JPG

labtastic

Posted 12 July 2016 - 09:51 AM

Probably supercoiled DNA. It is normal for intact, isolated plasmids to show multiple bands on a gel for this reason. 

 

If you linearize the plasmid by cutting in one place with a restriction enzyme, the supercoiled band should go away.

 

As a side note, it is generally not recommended to isolate plasmid from BL21 strains. They tend to be have a higher mutation rate and can be unstable (due to endogenous endonucleases carryover from miniprep). If you isolate the plasmid, transform right away into an appropriate cloning strain (e.g. TOP10), re-isolate the plasmid and quality control check by sequencing. To separate out the pLysS plasmid from your pET vector, take your miniprepped DNA, transform, and only plate on a single antibiotic. Your colonies are highly unlikely to have the second plasmid.