Hi,
I am currently working on to digest a plasmid using two different restriction enzymes. And I found out that the two REs that I chose were very close to each other and I have had trouble to verify if my digestion is actually successful.
I am using pNZ8149 plasmid, and used XbaI and SacI RE for the digestion, the difference between the XbaI and SacI on the plasmid was only 10bp. Therefore, I am unable to distinguish if my plasmid was actually being digested due to very small difference of band size when I did the gel electrophoresis.
I did sequential digestion since this what they recommend if the RE sites were close to each other.
I have attached the gel image for reference. Thank you in advance.
I would be a bit concerned because both of your single digests are showing two bands which suggests that neither enzyme alone completely digested the plasmid. You should have nice single bands in L2, L3 and L4.
Also why is there such a difference in brightness of the bands in the different samples, especially L2 and L3 compared to L1?
But you are never going to be able to see that size difference on a gel, which is why it is important to know that both single digests went to completion.
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