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Double digestion problem with PUCsp vector - (Apr/24/2015 )



 I am doing double digestion with AgeI and HindIII sites with PUCsp_COX(Choline oxidase) ~(2.6kb+ 1.6kb). 


I am keep getting a single band like the image attached below and it does not look like supercoiled DNA so I assumed that one of digestion worked.


The recipe of my digestion is


1st digestion(AgeI cut)


 3    microliter      NEB cutsmart buffer (100% activity with AgeI -HF(High Fidelity))


 3.3microgram    DNA(PUCsp_COX)


 sterile nano pure water to make total volume of 30 microliter


 1    microliter      AgeI- HF enzyme


Total 30microliter



 Digestion clean up with NEB PCR purification kit



2nd digestion(HindIII cut)



 same recipe as the first digestion with different digestion enzyme of HindIII-HF with NEB cutsmart buffer(100% activity)



**** please reply any possible reasons might interrupt with my result :)

Attached Image


You should not need to purify your sample between digestions.  You can add both enzymes (they have a compatible buffer!) and cut with both at the same time. But, one of them appears not to cut.  I don't know what the bands on your gel represent.  But you can set up a digestion with no enzyme, AgeI only, HindIII only, and both, and run all four samples on a gel. This will tell the story. You can also heat kill both of these enzymes, so you likely don't need to purify the DNA after your reaction, assuming you heat to 80 for 20 minutes. You can use a PCR cycler to do this after your 37 digestion.

You should likely cut down the amount of DNA you are cutting dramatically.  100 ng should be enough for most anything you need to do.  This could be your issue, since what you are seeing could really be incomplete digestion.