Problem with a Double digestion - ClaI/Esp3I digestion does not want to work! (Feb/22/2010 )

Hi there!

I am having some problems with a double digestion using ClaI/Esp3I

I just want to cut a piece of DNA from a plasmid, but something is not working. I know that the enzymes cut in my plasmid (when I do a single digestion I see the linearized plasmid). When I try another combination of enzymes, i.e. the enzyme "X" and one of the two enzymes involved I get a fragment, but when I use both enzymes I dont get a fragment, just the linearized plasmid!

So, in summary I see the same (linearized plasmid) if I do a single digestion with ClaI, or a single digestion with Esp3I or a double digestion with both of them! I use the buffer they recommend (Tango 1X from fermentas), at 37ºC plus DTT.

I tried 2h instead of 1h

I also tried a sequential digestion in both ways without success

And I tried using non-methylated DNA.

I need some new ideas!!!

If the cut sites are sufficiently close, you would not see two fragments after a double cut. If they are very very close, you may not actually cut with the second enzyme, since the overhang would be too short.

phage434 on Feb 22 2010, 03:31 PM said:

here a picture I just took (I did not run it more because I think is clear).
1- 1kb marker
2- 1ug of plasmid without enzyme
3- 1ug of plasmid + 1ul ClaI+ 1ul Esp3I in Tango 1X without DTT 2hours 37ºC
4- 1ug of plasmid + 1ul ClaI+ 1ul Esp3I in Tango 1X plus DTT 2hours 37ºC
5- 1ug of plasmid + 1ul ClaI 1h 37ºC. Then add 1ul Esp3I + DTT 1h 37ºC
6- 1ug of plasmid + 1ul Esp3I+DTT 1h 37ºC. Then add 1ul ClaI 1h 37ºC
7-100bp marker

I did not show the single digestions but they look exactly the same!!!!! Tomorrow I´m gonna start with the sequential digestions again! Any tips?? Should I do an overnight digestion??

How much DNA are you loading on the gel? Remember that the band intensity depends on the mass of the DNA, not on its molar concentration, so short bands will be quite a bit dimmer than long bands. But you still should see it, I would think.

Your gel shows that the plasmid is very large. Do you have a size estimate? You may be getting a band but not be able to see it because the amount of DNA in the band is small.

phage434 on Feb 22 2010, 06:35 PM said:

Well Phage434 thanks a lot for trying to help. I found what was the problem...I was using the wrong point mutation (thats what happen when you use plasmids with the wrong label ) , so the claI site was not where it should be; instead of 500bp there were only 20bp...

I just receive the new primers to produce the "real" point mutation, and I hope everything will work normally!!