My colleagues and I have an experience of successful cloning into various plasmid vectors. We noticed that it is hard to clone some inserts into one vectors, while the same inserts can be readily cloned into other vectors. In our hands, one of these hard-to-clone vectors is pEGFP-N1. With various cloning strategies my colleagues and I obtain a very few colonies, which often do not contain the vector with the insert. The same inserts can be easily cloned in other vectors. What can be wrong with pEGFP-N1? Did you have the same problems with some vectors? Did you solve similar problems?
Hi Dmitry,
this vector is famous in our lab, because It happend several times that people didn't get colonies.
Since it carries an KANAMYCIN restence istead of ampicillin which is the standard selection marker of most cloning vectors used in our lab. 50 µg/ml final concentration works fine for selection on agar plates.
I hope this simple solution helps you.
http://www.human.cornell.edu/dns/qilab/images/pEGFPN11_1.jpg
Best Kai
Thanks, Kai. Indeed, we sometimes make a mistake and use ampicillin plates instead of kanamycin plates. The strange thing is that even on right kanamycin plates we get a few colonies. Is it possible, that during plasmid manipulations we purified a vector that has some toxic properties for E.coli? The matter is not in sites for restriction endonucleases since plasmid is cut by them.
As I know pEGFP-N1 contains CpG islands can they somehow influence cloning process?
Hi Dmitry,
In literature has been described specific GFP protein for bacterial that differ in some amino acid. I think that this two protein differ because the mammalian GFP is toxic for bacterial.
I think that probably your insert could have a potential role in gene expression (i.e promoter) so you have the GFP expression in bacterial. I tried many time to work with the EGFP (from pEGFP-N1) under bacterial promoter with your similar results.
I tried, also, to clone e similar insert (in bp and restriction site) to verify the backbone quality and I obtained colonies easily!
Good luck
Thanks Antonio. Very interesting! I think bacteria can express almost any protein from plasmids, no matter what kind of promoter is used. Perhaps it is due to non-specific transcription or transcription noise. Could you provide the link to the article describing bacteria-specific GFP?
Hi Dmitry,
I do a simple consideration: if you compare the aa sequences between EGFP and common plasmid used for GFP expression in bacterial (see ADDGENE for a complete list of plasmids) you'll look a difference in 3/4 amino acids position.
I apologize but today I don't remember the article in which is described it but surely there is a problem in the protein folding.
You could look here: http://www.nature.com/nbt/journal/v24/n1/full/nbt1172.html
The best
Antonio
So, our problem was that we do not outgrow transformed cells. About 30-45 minutes of growth on SOC medium is enough to obtain large quantity of colonies.
- Badges
- Science topic