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DNA digestion - Restriction Enzymes (Mar/19/2009 )

Hye
I have a problem with the digestion of genomic DNA of bacteria Bacillus with NotI and SfiI. I didnt find bandes of restriction after an over night digestion in 37°C. I use elctrophoresis migration under agarose gel 1.2%. If some one have done this experience, could he help me. Please I'm really in destress. Thank you a lot :(

-emna-

Use your notI and SfiI to cut some other DNA to check if they are working. Try cutting your genomic DNA with another coommon enzyme like ecoRI or NlaIV etc. This will show if your DNA is okay or not. Otherwise, try purifying your DNA by a column or precipitation and try again.

-lotus-

These are 8 base cutters, and will almost always produce long fragments after digestion. A 1.5% agarose gel has no hope whatsoever of resolving these long fragments. You would need to do a pulse field gel (or at least a field inversion gel) to resolve these fragments successfully. I would guess that your digestions worked, but that you can't see the results. You may very well need to modify the DNA prep if you want to resolve fragments of this size. What is the end goal?

-phage434-

phage434 on Mar 20 2009, 02:46 AM said:

These are 8 base cutters, and will almost always produce long fragments after digestion. A 1.5% agarose gel has no hope whatsoever of resolving these long fragments. You would need to do a pulse field gel (or at least a field inversion gel) to resolve these fragments successfully. I would guess that your digestions worked, but that you can't see the results. You may very well need to modify the DNA prep if you want to resolve fragments of this size. What is the end goal?

Thank you for your answer, I'm doing this digestion to look for a fragment containing a gene which I will screen using southern bot. I have forgetten to add that I have obtained only one a bande in agarose gel with NotI and a smear with SfiI. :lol:

-emna-