Why can I see no band on gel after restriction digestion?

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bio.nesliii
bio.nesliii's picture
Why can I see no band on gel after restriction digestion?

 Hello,

I’ve a problem about restriction enzyme digestion of plasmid. I add 1X buffer,1ug plasmid,10u enzyme and distile water (Total volume is 20 uL). I check the gel for plasmid but, I can't see any band in gel. I don't know Why? Do you have any suggestions for me please? Thanks a lot!

Jason King
Jason King's picture
 Can you see any bands on the

 Can you see any bands on the gel? Ie. marker lane and the vector from which the insert was supposed to have been cut.

if you see nothing then either:
1) there's no ethidium bromide or alternative DNA stain present
2)the buffer used to run the gel is wrong and the gel hasn't run
3) the gel ran in the wrong direction so your samples ended up n the buffer

if you can only see one band on the gel and not two then most likely one of the cutting sites is not as you expected. Is the size of the plasmid correct? Very often, if you've obtained the plasmid from a non-commercial source it turns out to be something else completely.

If you supply a bit more information then we might be able to help further.

PM