Hello Dear,

Im PhD student

I was using Gibsson assembly, and I cloned my gene onto the Psl18 plasmid . The plasmid was right when I sent it to Sequencing after geting a positives colonies.

However, when attempting to perform PCR and amplify my gene using my glyceral bacterial stock, I obtain a bande that is appropriately sized, but another bande that is nearly the same size as my gene (see gel picture below).

I don't understand why, as I should only have one bande! Can my plasmid exist in two different forms in the Glycrio Stokc 9 (one that is correct and the other that is not) or is there another explanation?

Or should I do the cloning all over again ?

It`s shloud be a primers issues ! (Althought I checked it in snapgene but it`s bande just in my gene of interest)

Also, I test 2 taq (polymerase and Q5 HF) but I got the same results.

I appreciate your help in advance.

Thanks

Ayoub