Hello!
Recently, I ordered an FOLH1 ORF, which came inserted into the pUC-57 mini vector. Additionally, I requested BamHI and XbaI restriction sites to be included at each end of the ORF. When I performed the digestion of the plasmid, I observed that the single digestions with BamHI and XbaI linearized the plasmid as expected. However, when I performed the double digestion, I noticed two bands corresponding to the pUC vector and the FOLH1 ORF, as anticipated. Surprisingly, there was an additional band below them that I did not expect. What does this additional band signify? Could it be contamination, or is there a problem with the digestion process?
Hello Paula Iglesias Matesanz ,
what are the size of the band you are expecting (pUC back bone and FOLH1 orf insert ) ?
Even if your single digestion lane show only the linearized vector, still check the pUC for pre-existing BamH1 or Xba1 restriction sites.
Do you have another enzyme/restriction site at your dispoal to do another double digestion and si if the problem comes from any of your enzymes ?
Good luck!
Philippe.
The pUC should be around 1800bp and the FOLH1 gene is 2253bp.
I already checked that both BamHI and XbaI don´t cut the vector. I added the restriction sites to the ORF.
Yeah, maybe I can try to see if other enzymes cut properly. But do you think that this additional band could be because the enzymes aren´t working properly?
Thank you for you answer!
Hi Paula Iglesias Matesanz ,
some enzyme can have "star" activity where they cut to other stites than the regular one.
It can happen in specific conditions (for example to much glycerol in the reaction mix) or if the digesion is too long.
https://www.neb.com/en/tools-and-resources/usage-guidelines/star-activity
What king of enzyme are you using ? High fidelity from NEB ? and the buffer ?
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