I am trying to clone an insert ~1.8kb into a vector of ~18kb using electrocompetent e.coli cells. The last attempt was only partly successful as although most of my insert appeared to be there, there also seemed to be the tail end of it missing. I don't understand how only part of an insert can be cloned? Can someone explain this to me?
Hi Lemon,
Which Restriction enzyme sites you use? Please verify for an extra restriction site at one end (most probable). If the clone is sequenced check the cloning sites?
Other possibility is star activity of the enzyme (theoretical, I have never encountered)
Best
Is that your gene contains two restriction site for the same enzyme? Yes there might be possibility that your restriction enzyme is showing start activity or maybe you did over-digestion of your gene of interest.
You can check this on this website NEB cutter 2.0
http://nc2.neb.com/NEBcutter2/
There you can check if there are more than one restriction sites for enzyme or enzyme combination you are using. You can also reduce Glycerol concentration, read the content below for more information:
https://www.neb.com/faqs/1/01/01/what-is-star-activity-and-how-can-it-be-avoided
Hope it helps!
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression