Hello all,
I am doing point mutation using overlap methods. I got my overlap , transformed to vector got positive colony pcr result as well. Isolated the plasmids again did pcr with the plasmids got positive result then sent the same plasmids for sequencing. The alignment from the sequencing result is scattered. After seeing the results I again did pcr with the same plasmid and I can see positive result. 4-5 times I did this and got false sequencing result.
What could be the reason for false sequencing result? Should I go ahead with expressions and purification with this plasmid without confirming through sequencing?
Primers can sometimes amplify contaminating sequences. I would go back to your first generation of plasmid and sequence that.
Can you share a picture of the sequencing alignments? I'd hesitate to move forward with experiments prior to confirming the DNA change. Any chance you can do a diagnostic digest to help check prior to sequencing?
Amy Klocko .
I did insert release but it was a very smear insert release.
Have attached the alignment image
That’s a very poor match in the alignment, is that your insert? Either the sequence quality is poor or the DNA isn’t a match, I’m leaning towards the sequence quality being poor. I would prep up fresh DNA and retry the sequence but send 3-6 preps to have extras. Maybe do an extra wash step first to remove excess salts too. Good luck!
- Badges
- Science topic
- Similar topics
- Filing