cloning mystery - numbers are perfect, but no insert (Feb/28/2007 )
Hello all,
Another mystery, maybe that's something obvious but I can't solve it by myself...
Here's the deal : I'm trying to clone PCR amplified fragments in a vector. I ordered oligos with restriction sites on their end (XbaI and HindIII). I got PCR fragment, I cut them, purified them on a PCR cleanup column. I cut the vector with the same enzymes, CIPed it, gel purified it. I set up 4 ligation, 3 in order to clone my 3 inserts, and 1 as a control of vector alone without any insert. After transformation, I got several hundreds of colonies for my 3 actual ligations, and something like 5 colonies for the control ligation. So everything looked perfect. Problem is : when I minipreped colonies, I never found any insert inserted in the vector (I use the same XbaI and HindIII enzymes to check the plasmids by restriction).
Any idea or suggestion about what might have happened?
Thanks!
Another mystery, maybe that's something obvious but I can't solve it by myself...
Here's the deal : I'm trying to clone PCR amplified fragments in a vector. I ordered oligos with restriction sites on their end (XbaI and HindIII). I got PCR fragment, I cut them, purified them on a PCR cleanup column. I cut the vector with the same enzymes, CIPed it, gel purified it. I set up 4 ligation, 3 in order to clone my 3 inserts, and 1 as a control of vector alone without any insert. After transformation, I got several hundreds of colonies for my 3 actual ligations, and something like 5 colonies for the control ligation. So everything looked perfect. Problem is : when I minipreped colonies, I never found any insert inserted in the vector (I use the same XbaI and HindIII enzymes to check the plasmids by restriction).
Any idea or suggestion about what might have happened?
Thanks!
how much DNA did u digest from the miniprep? Bcoz if one used a less DNA for digestion, one may or may not see the insert on gel. try to use more DNA and digest it.
I am just guessing this as the control has only 5 colonies compared to the ligation ones.
Or run the uncut minipreps along with the uncut plasmid, assuming the original plamid doesnt have any insert. If there is a shift in the plasmids from miniprep, then u sure do have the inserts.
Good Luck !!!
How long is your insert?
Did you run on a gel your minipreps and your vector without insert? If you have insert in your clones, you should see they run a little slower than the vector. If they do, It’s almost sure they have the insert.
When you digest your minipreps, you said you didn’t found any insert. But, can you see the vector in its linear form??? Because may be your digestions are not working. Why you don’t digest with an enzyme that cut the vector (one or twice).
Mystery solved!
I managed to design my oligos in a way that created a perfect methylation site right to the XbaI restriction site, and of course I'm not using a dam- strain...
So the insert was there, the only problem was that I could not take it out using XbaI again...
Thanks for your suggestions anyway!