Nde1 works on positive control, not on DNA sample - Restriction Digest (Jul/20/2007 )
I was wondering if anyone had advice on troubleshooting with Nde1. I have been trying for two weeks to get my restriction digest to work on genomic DNA. Using lambda as a positive control, the digest works decently (still not great), but does not appear to work at all on my DNA sample. I have adjusted DNA, buffer, BSA, and Nde1 concentrations.
I use a 15-hour cycle of 35 C with 20 minute 65 C inactivation. The original protocol called for 500ng DNA, 1ul (20 units) Nde1, 12ul buffer (NEB4), and 12ul 1/100 BSA (water to 120ul). I have even tried Promega versus New England BioLabs Nde1.
If anybody has any advice, or has had similar problems, I would appreciate any feedback. Thanks!
I am not sure if NdeI is methylation sensitive restriction enzyme or not, if it is it will be sensitive to some specific sequence. I have ever used XbaI , it can cut my other plasmid but can not cut the XbaI site followed by TC, I got the free strain of DAM- from NEN and transfect my clone into it,then it can be cut. check NEB website about the methylation information if NdeI is a methylation sensitve enzyme in the instruction book.
I guess the BSA is 1.2ul not 12ul (then it will be 10x). Just verify the vol. of BSA. BSA comes as 100x.
You didn't say where this genomic DNA came from, but every restriction enzyme will fail to cut genomic DNA from a species which expresses the gene for that enzyme. It is not unusual to find that an enzyme tried on genomic DNA from an novel species fails to cut it.
how long you digest your genomic DNA with NdeI???
genomic DNA need overnight digestion
Thanks to everyone for the responses. For starters, Nde1 is not methylation sensitive, so that should not be a problem. I originally tried a 4-hour digestion, but quickly changed to an overnight (15 hour) digestion. I have tried using BSA at both 1/100 and 1/10 dilutions - both worked on the positive control, neither were effective with my DNA sample. I am working with bat DNA from the genus Myotis; my next planned move is to try a different restriction enzyme with similar cutting properties as Nde1, so perhaps that is the best option.
NdeI is not methylation sensitive to DNA from E. coli. This is definitely NOT true in general. In particular, it is sensitive to N6 methylation of the second A in the sequence CATATG. See Rebase and Gene 74:43-44, Silber 1988.