daisato

I have cloned a 500bp insert into a 7000bp vector, and I'm trying to confirm for correct insertion by use of a restriction digestion. Per sample I've done three reactions:
- uncut control
- digestion with AscI and NotI, which cut on both sides of the insert to check for insertion of the 500bp insert
- digestion with AscI, NotI and SmaI, which cuts twice in the insert to check whether it is the correct fragment that has been inserted.


The setup for the reactions was:
- 2.5ul 10x buffer
- 0.25ul BSA
- 0.5ul enzyme, 10U
- 500ng template DNA
- up to 20ul water.

Digestion at 37C for over an hour.

The problem is that when I run all three reactions on an agarose gel after digestion, I don't see any product at all. Not the expected digested bands, but also no undigested fragment. Also the uncut control is empty; ergo, all lanes are completely empty. Quantification by nanodrop reveals sufficient amounts of DNA in all samples, so I would expect to see at least some uncut DNA

Could anybody give me a suggestion on how to deal with this problem? If there is more information required, I will happily give it.
Thanks a lot in advance!