How to get an individual plasmid out of a plasmid mix in E.coli - (May/11/2008 )
Hey,
recently I isolated DNA fragments from a gel and cloned them into a vector. I know that the DNA which I isolated from the gel contains several different fragments of the same size. Hence, my ligation reaction will contain my vector with different fragments. I then transformed my ligation into E.coli.
I tested all colonies via PCR for the plasmid I wanted. However, almost all of my colonies showed a band on my gel. Which was surprising at first because I didn't expect more than one positive clone.
I assume that each E.coli cell received more than only one plasmid. So each cell has a mix of all different plasmids. Now I was wondering if anybody knows a way to get a clone that only contains the one plasmid that I want.
Thanks
Benjamin
Thanks
Benjamin
1. Yes, a cell may receive and amplify more than one plasmid with similar ori of replication (and sometimes dissimilar too, but less likely).
2. You do the repeat round of transformation using a very low amount of DNA from your first round. That will result in each bacteria getting only one plasmid, and then your colony screen will give you pure clone. It almost always works, but you should be prepared for one more round.
3. But there is also a chance that you may be seeing PCR contamination, use controls to make sure that is not the case. If this continues to be a problem, do colony blot and slect the brightest clones for relative purity.
HTH/
Thanks for the quick reply.
I am pretty sure that I don't have PCR contaminations since I am running positive and negative controls.
But how can I be sure that even if I use less DNA that each cell gets only one plasmid. How much is much less? Like one nanogram?
It's actually rare to get more than one plasmid type in a cell (without strong selection for both). Typical transformation efficiencies are quite low, and plasmid segregation with no selection bias eventually pushes to a single plasmid type in each cell. This happens quickly for low copy number plasmids, and very very slowly for high copy number ones. If you're worried or find this is a problem, use a low copy number plasmid. But I'd worry about this when I see it, not before. Miniprep and sequence a few of your clones and see if there really is a problem, or if you are being confused by colony PCR results.
As Phage said, two clones in the one cell is rare. Virtually all your clones will contain one clone. Maybe the reason for your unexpected high percentage of positives is that the insert you screened for was the major fragment in your ligation mix hence it is in the majority of your clones. PCR screening can also produce false positive results quite easily due to excess ligation mix outside your E. coli acting as template. Screening by miniprep and digest will confirm the identity of those clones. I'm assuming you used a vector and insert primer so the PCR was specific for your insert.
By the way, if you use ultra-competent cells, some cells may recieve more than one plasmid, and so some colonies may have mixture, but as you say, all colonies that you screened gave more than one, it is next to impossible. So, I would focus more on your screening strategy, pcr or bacterial colony contamination issues etc.
Thanks guys, I think I will just go ahead and sequence it. See what that gives me.