Qiagen DNA Kits - Extra bands (Nov/07/2006 )
I have a question about Qiagens DNA prep kits and clean up kits. We quite often end up with an extra band running below our DNA of interest, when using a kit from Medicorp we do not get this band. Have you any ideas where this extra band is coming from and should we be concerned?
what is your starting material, are you extracting plasmid or genomic or PCR product, which kit are you using?
I am trying to clean up a PCR fragment and then the subsequent digest of this fragment. I cut out the band of interest, use the gel extraction kit, elute, digest, clean up again to switch buffers, do a second digest, run this out on a gel, again cut out the 1600bp band, use the gel extraction kit. I then run out a uL to look at the concentration for cloning and there is a second band running lower then the 1600bp band??????????
what is your starting material, are you extracting plasmid or genomic or PCR product, which kit are you using?
I am trying to clean up a PCR fragment and then the subsequent digest of this fragment. I cut out the band of interest, use the gel extraction kit, elute, digest, clean up again to switch buffers, do a second digest, run this out on a gel, again cut out the 1600bp band, use the gel extraction kit. I then run out a uL to look at the concentration for cloning and there is a second band running lower then the 1600bp band??????????
When you do a restriction digest, any smaller fragment will be present in the band corresponding to the larger fragment.
It sounds crazy, but it's true. The smaller fragment will run in the gel cleanly, the larger fragment will be contaminated with the smaller fragment.
Is the lower band the same size as one of the undesireable fragments from the previous digestion?
what is your starting material, are you extracting plasmid or genomic or PCR product, which kit are you using?
I am trying to clean up a PCR fragment and then the subsequent digest of this fragment. I cut out the band of interest, use the gel extraction kit, elute, digest, clean up again to switch buffers, do a second digest, run this out on a gel, again cut out the 1600bp band, use the gel extraction kit. I then run out a uL to look at the concentration for cloning and there is a second band running lower then the 1600bp band??????????
When you do a restriction digest, any smaller fragment will be present in the band corresponding to the larger fragment.
It sounds crazy, but it's true. The smaller fragment will run in the gel cleanly, the larger fragment will be contaminated with the smaller fragment.
Is the lower band the same size as one of the undesireable fragments from the previous digestion?
All I am digesting is the ends (6bps) of my PCR fragment.
Well, I can't explain it then.
We see extra bands when we plate clean our PCR product, and have only gotten as far as learning that it is important to use buffer (not water) to resuspend the DNA in. We don't know why this would make a difference, but it is as far as we have gotten on it.
Are you eluting with buffer, or with water?
Sorry I can't be of more help.
may you try to heat little your DNA to 65° before loading? I'm thinkg of a partialrefolding of your DNA fragment which happened me once with a shorter (300bp) fragment that gave me 300 and roughly 550 bp 
the upper band disappeared after quick heating (in loadin buffer and direct loading after)
We see extra bands when we plate clean our PCR product, and have only gotten as far as learning that it is important to use buffer (not water) to resuspend the DNA in. We don't know why this would make a difference, but it is as far as we have gotten on it.
Are you eluting with buffer, or with water?
Sorry I can't be of more help.
I have been eluting in water, I could try buffer,
Thanks