Digestion bands appear faint - (Oct/22/2014 )

Hi, I am having a problem with visualizing my restriction digestion products; beside that they sometimes show up uneven they become faint and some bands won't be cut given that the least number per well I must get is two and a maximum of four.

What could be the reason/s?

More details needed - what are you digesting, how big are the fragments, how much DNA are you digesting...

bob1 on Wed Oct 22 20:02:47 2014 said:

More details needed - what are you digesting, how big are the fragments, how much DNA are you digesting...

I am digesting a 740 pb PCR product (VDR)

For the restriction I put in a tube: 3.5 ul PCR products, 1ul provided buffer, 0.5 Taq I restriction enzyme, and 5 ul nuclease-free water (I use New England Biolabs CutSmart restriction enzyme with its buffer)

I incubate for 35 minutes at 65 oC then run them in 2% gel

The expected bands are:

TT-> 245 and 495

Tt-> 495, 205, 245, and 290

tt-> 205, 245, and 290

P.S. I don't know how much DNA is in my sample but when I get a slightly faint PCR band I use 5 ul PCR products, 1ul provided buffer, 0.5 Taq I restriction enzyme, and 3.5 ul nuclease-free water

I would use more DNA. Your shorter bands will be fainter simply because they have smaller amounts of DNA. The ladders show up well, so you simply have insufficient DNA. Can you add more than 10 ul to the well? If so, digest in 20-30 ul (whatever the maximum amount you can put in the well) and add more PCR product. You should also clean the camera platform, since there is quite a bit of background junk.