I am trying to clone my gene in pet22b (using NdeI/XhoI double digestion for 4h, gel extraction and ligation at 16 deg overnight). I got a couple of positive clones in colony PCR, when I extracted the plasmid from them, there were 2 bands, one same as the original vector and other one larger in size by 1kb (corresponding to my gene).
I'll be confirming by restriction digestion tomorrow. I just wanted to know if someone has observed similar phenomenon during cloning. Is it good enough to be used as a mixture to study protein expression or should I gel-extract the recombinant plasmid and transform again?
1) It is possible that the colony you selected for the plasmid preparation was not a colony from a single colony forming unit (CFU) probably you need to spread the suspension better. It can happen that two cells (one with the correct plasmid and one with self-ligated plasmid) were very close to each other after plating and then they grow into one convergent colony. So you end up with two kind of plasmids from this prep.
2) Or it can be that while selecting a colony you somehow selected not one but two colonies since they were very close to each other.
3) And the last possibility is that you have the correct plasmid, the smaller band you see could be the covalently closed circular supercoiled plasmid that will migrate faster in a gel, whereas the band you see at the correct size could be the linearized plasmid, the digestion will clear up this issue.
Hi
It is also possible that the clone you have selected may be unstable and is loosing the insert....this problem is often observed .....and if it is true after some time your clone will not have the insert......the only way is to screen more clones
bye
Will a problem of unstable clone get solved if I extract the recombinant plasmid and use it to transform again, and select new clones?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning