do the sizes of the xho bands without the extra one add up to the correct goi size?

If so the extra band comes from the plasmid. Can you cut a non insert plasmid sample to see if it generates the extra band which does look very faint so is probably contamination ?

Jj Goh

(1) I agree with Paul Rutland , check a digestion on non-insert plasmid as a control. I usually do that. (2) I checked online, the vector you are using is In-Fusion®-ready linearized adenoviral vector for constitutive expression of a gene together with the fluorescent protein ZsGreen1. Since it is used to express your gene, to make sure no base mutations on it, you should also check the inserted sequence of your GOI. Sequencing should start from a little bit outside the insert (using backbone sequencing primer), containing some backbone. If the sequence orientation is correct, your gene sequence is correct, then it is probably ok to go ahead with your next experiment.

Thanks a lot Paul Rutland & Yuan-Yeu Yau for the replies. I did not try with non insert plasmid as the XhoI digestion for pAdenoX-ZsGreen1 which inserted with the control fragment showed good results with no extra band.

However, the sequence results for the inserted GOI showed no mutations.

Wondering if there is a possibility that the extra band was contamination of the E coli genome? Because after the cloning, I expanded the plasmids through bacterial culture, then followed by plasmid purification before RE digestion.

I do not think that the ecoli genome idea is right Jj Goh . Genomes are large and complex and when you cut with a restriction enzyme produce many bands which all merge into a smear of cut dna not a single band which is visible as one size. A single band suggests that it is related to amplified material which would be via the plasmid I think but it does make me wonder where the changed NHE band is seen because that digest looks clean. It is a shame that the band is so faint because sequencing would clarify where it came from. I appreciate that the goi sequencing was good but if the odd band is outside the sequencing primers it would not sequence. Was the sequencing done with gene specific or plasmid specific primers outside of the goi?

Thank Paul Rutland for the reply. The sequencing done covered part of the backbone and full length of the GOI. As the next step of experiment will be transfection of HEK293 cells to package the adenovirus, wondering if there is no mutation in the GOI, it should be fine to proceed accordingly?

Back to the suggestion of digest the non insert plasmid, it might be difficult as the adenoviral vector (linearised) comes in only 10ul and digestion will use up 5ul. Also, due to the large size of the plasmid, it is not recommended to keep glycerol stock of it. In addition, the plasmid is not sold separately and the whole kit is rather costly.

I would expect the transfection to work well with the goi sequencing well but Yuan-Yeu Yau has more experience than I do in this aspect so hopefully he will comment further and agree that the control part of the experiment would be very expensive

Jj Goh I have though about that. When we did the control digestion, we had plenty of miniprepped backbone vector. So, we have enough material to spend. Yours are linear commercial product. It would be costly to do the similar control digestion. I would just go ahead do the transfection of HEK293 cells, and see what the results turn out.

If you really want to find out that extra band, you probably can circularize the plasmid (with a small amount of the commercial product), then do a transformation and miniprep to increase the amount of the vector for empty vector digestion?! But I am not sure if it is worth the time? or your advisor will allow you?

Either there are contaminations in dna plasmid or the restriction enzyme cuts extra region in plasmid or even it is cut in unproper region in plasimid

Thanks a lot Paul Rutland & Yuan-Yeu Yau for the replies. I shall discuss with my advisor then.

Thanks Tareq Al-Omari for your reply too.

If you really want to know what the band is, then you can digest a large amount of your GOI:adeno vector plasmid, then gel extract the extra band. Ligate the extra band to a plasmid with know sequencing primers and sequence.

SInce your GOI sequencing showed no mutation then you can try to make the adeno virus, infect your cells and see if you get expression of your POI. if you get expression then no need to worry. If you do not get expression then maybe your vector has recombined somehow and this recombination affected sequences in the plasmid that are important for transcription or translation of your GOI.