I am trying to express mammalian protein in bacterial system. Protein Size 70kDa, vector backbone pET28a+. I received the gene in pUC backbone, when transformed in DH5a, showed clonal variation. Upon checking, correct clone was selected and assumed that after ligation, there might not be any such difference but to my surprise, there also i am getting clonal variation. I have taken negative controls like vector, insert and competent cells to negate any extraction and handling errors. Would appreciate suggestions and method to understand this problem.
when i am picking four different colonies from the same plate. 3 shows correct cut sites and one shows wrong cut sites
Sometimes there is a selection against plasmids with high level of expression. So it might not be a surprise that you pick up variants that have various deletions. Just pick one or two that look correct, test for expression or sequence, and proceed.
Thank you sir, I will proceed forward. what i was concerned was whether such changes might occur later during the expression stage? and i could not understand what does selection against plasmid with high level expression means. After ligation, either i would get ligated vector, uncut vector or uncut insert. Since, i checked with vector and insert control, i dont know why such variation? Are this deletions occuring due to large gene size?
Although you see a clean PCR product, it can be that unespecific amplifications are cloned in your plasmid giving you that variability. Probably you have done it but cuttinig the DNA band out of the gel is safer. pET28a is a stable plasmid, I would not think that something is wrong with your plasmid but your cloned fragment.
Even if you see a clean PCR product, it can be that unespecific amplifications are cloned in your plasmid giving you that variability. Probably you have done it but cuttinig the DNA band out of the gel is safer. pET28a is a stable plasmid, I would not think that something is wrong with your plasmid but your cloned fragment.
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