Dear professors and colleagues
I would like to get your opinion on the following figure:
1- I made a ligation for the specific 200bp insert to vector and that exact location shows misreading in 3 different oligo primers and identified by 1 oligo primer. What is your opinion?
2- In this case, does it mean that the insert is not right?
Thank you in advance
Similar results can be caused by salt, sample overloading ,too high a running voltage and most often by old and dirty capillaries. Can you add the original sequence (.abi) file for the one that worked and one of the fail sequences as there may be clues in the running parameters embedded in those files
Have you verified gene cloning by restriction digestion and PCR? If yes, did you get positive results? If yes, then there might be an issue with the quality of the recombinant vector that is used for sequencing or poor handling of the person who is performing sequencing.
Good wishes!
- Badges
- Science method