I have used a vector of 3.8kb and my insert is of size ~370bp. I have confirmed the vector size with single digestion before proceeding with the cloning process.
I have isolated the plasmid from transformants and first confirmed the insert via diagnostic PCR. Then those were digested with BamHI and XhoI. If my insert is present then I should get a popout of ~600bp and backbone of ~3600bp. Here Iam getting the popout with a good intensity at the desired size. However, the backbone band appears to be very faint at the desired location. Here Iam attaching the image.
Iam not able to figure whether these are positive transformants or not. If yes why am I not able to get intense band for the backbone after digestion.
I would assume that the strong signal at 6kb is the vector and,as you say, is a strong signal compared to the insert because the longer vector traps more ethidium bromide so looks brighter. The question is why the vector is running at a higher size than expected. It might be worth a restriction digest of the strong band with an enzyme that cuts often and see what the size of the band is when its smaller restriction products are added together to see if this is just a gel running or secondary structure artifact
I concur with Paul Rutland that the 6kb band is most likely the vector (note it is also present in your vector only). Maybe you inadvertently created a vector dimer, or maybe you have background from the source plasmid of the insert?
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