Hello everyone,
I have a recombinant plasmid always getting overlapped peaks when sequencing. The sequencing primer is located on the backbone of the vector, tens of nts ahead the cloning site, and the primer works perfectly for other plasmid using the same vector.
I have tried purifying the plasmid by plate streaking which turned out no help.
What's weired is that the plasmid expresses the correct proteins.
So, I wonder if it is possible that the E.coli contains 2 different sequences that I can't get it purified by plate streaking? However, this is contrary to the plasmid incompatibility theory.
Do you have any suggestions? Thank you!
It is possible that two plasmids co-exist in the cell, so restreaking will not solve it. My suggestion is to dilute a little of your DNA and retransform it at very low DNA concentrations and then try a few of those colonies. It could be they are nearly identical but maybe slightly frame shifted, or you have some vector alone contaminating the actual clone.
The other possibility is that there is some secondary structure in your plasmid so you are getting some frame shifted sequence after the secondary structure.
It's possible that your inserted gene contains some sequence that is similar enough that the primer is binding to it and reading downstream of where you expect. It's sometimes hard to do, but can you try to decipher a portion of the overlapped sequencing read and then search your plasmid for that sequence. Set your program to allow a few mismatches and also search for the primer sequence allowing mismatches to see if there are obvious similar sequences where the primer may have bound.
- Badges
- Science topic
- Similar topics
- Vectorization