Recently，I am constructing a plasmid which insert a yeast gene . When sequencing always the same sequences (about 1500kb)from E.coli replace my target gene partial (800bp).At first ,I think my template plasmid was contaminated ,so I used yeast genome as the template.Then , similar sequencing result was sent to me.
whether I the yeast genome also contaminated ?Or my primer is not specific enough?
Maybe when I extract the plasmid from DH5α too fiercely so that always replaced with E.coli genome?But it is reasonable to insert the same nucleotides in the same place?
Thank you for your attention.
I do not know about the 2 genomes producing this effest but have you checked that there is no post pcr contamination of you ecoli gene in your working area or in the pipettes. Do you run "no template controls" in your pcr reactions to eliminate the possibility of contamination?
I would agree with Paul Rutland that you have some contamination somewhere. It could be you have E. coli DNA contaminating things, or it could be that the plasmid with the E. coli gene is present in the lab and contaminating things. Can you design primers that are highly specific to the yeast gene and not the E. coli gene, they should be sufficiently different.
You could also sequence your PCR product to see if that is actually correct.
I would first carefully analyze your primers again to be sure there is not a mistake in them, and determine their specificity for the two genes. If need be, then order new primers (much cheaper than sequencing). Be sure to run the right controls as mentioned above.
Thank you，Paul Rutland & Michael J. Benedik . I seldom set a negative control in my pcr reactions . I'll give your suggestion my careful consideration.
Currently，maybe the primer I used in sequencing not specific enough.The first company sequence results were polished and didn't noted the overlap peak in the reaction.
I've ordered a new and more specific primer to used in sequencing.
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