Plasmid transformation - (Feb/08/2008 )
Can a mini prep that was prepared from a culture originated by one single colonie contain a mixture of plasmids?
Being reductive, can 2 plasmids be transformed to one bacteria?
If they are self-compatible they can co-exist in the same cell.
I'll be more specific. Sorry I wasn't precise first time.
I'm doing mutagenesis. I transform the reaction to super competent e coli. I plate them and next day I pick up several single colonies to liquid LB, off course each in a different tube, and performed a mini-prep. I had some strange result from sequencing, so I wonder if a mutated and a non mutated can be transform to the same bacteria, or better, same colonie can have a mix of plasmids.
Thanks
I'll be more specific. Sorry I wasn't precise first time.
I'm doing mutagenesis. I transform the reaction to super competent e coli. I plate them and next day I pick up several single colonies to liquid LB, off course each in a different tube, and performed a mini-prep. I had some strange result from sequencing, so I wonder if a mutated and a non mutated can be transform to the same bacteria, or better, same colonie can have a mix of plasmids.
Thanks
I think that usually only one plasmid enters a single cell, but if your colonies were growing close together then its possible that you picked up some cells from a neighbor in which case you could have two types of plasmids growing in your liquid culture. If you're concerned about it, then you can streak your colonies onto a new plate to get individual colonies again. Does your sequence read look like its two in one? maybe your primer is annealing to two different sites that are very similar, in which case you might want to try a read with a different primer.
We had a very strange case which there was a clone having two back-to-back copies of the same plasmid, one with a mutated origin, such that it was inactive. When we sequenced it, everything seemed normal except that the origin looked as if two different clones were present in the cell. We streaked for single colonies, retransformed, sequenced, and did restriction digests, and they all looked fine, except for the sequencing in the ori. Finally, we cut with an enzyme, religated, retransformed, and eliminated the problem, but strange things can happen.