Hi. I have faced a problem I never seen before. I have Origene pCMV-XL6 Entry plasmid without any ORF - just empty plasmid. It has Kanamycin resistance so I transform plasmid in DH5a E. coli cells, select colony and grow in in 200 ml LB media with 25 ug/ml Kanamycin ON in 37 degrees. Then I use Maxiprep from Origene or Qiagene to purify plasmid. For quality control I run 1 ug plasmid on 0.8% Agarose gel. My problem is that several Maxipreps I got different size plasmids. In some I got ~6K bp plasmid (super-coiled) and trace of 2 bigger size (partially relaxed and relaxed) plasmids. But sometime I got only relaxed plasmid, no super-coiled. I tried to use restriction enzymes - but they showed just my plasmid is relaxed. Does anyone have any suggestions how to get super-coiled plasmids only?
In my experience batches of plasmids done in different times with different columns tend to vary according to the fraction of supercoil DNA. The first thing is to review the protocol and your manipulation as you may be introducing nicks that could increase the fraction of slow migrating plasmids.
If you want to work exclusively with supercoil DNA I suggest the old-school technique of cesium chloride gradient ultracentrifugation (https://www.youtube.com/watch?v=n-lVxNjfeio). And here the ref for the method: https://www.thermofisher.com.au/Uploads/file/Scientific/Applications/Equipment-Furniture/Traditional-Methods-for-CsCl-Isolation-of-Plasmid-DNA-by-Ultracentrifugation.PDF
Depending on how pure you want, you may need to do a cesium gradient isolation twice (After isolating the fraction, reload on a fresh gradient and repeat the centrifugation).
Important trick: when puncturing the centrifuge tube with a syringe to extract the band it is common to start leaking material. Placing a magic (adhesive) tape on the spot where you are going to insert the needle helps prevent leaks and spills. Wear gloves and masks as it uses lots of ethidium bromide. Good luck.
I concur with Alvaro that if you truly need supercoiled plasmid then a CsCl gradient is the way to go. However for most applications it does not matter. Is there a reason you need supercoiled?
Hi Dr. Smagris! Can I asked how do you use restriction enzymes to show your plasmid is relaxed? I have this issue as well, but I found sometimes the enzymes are able to cut at the right sites even though the plasmids are relaxed.
- Badges
- Science topic