Restriction digestion and DNA degradation problem - Restriction digestion and DNA degradation problem (Oct/07/2007 )

Hi all,

For the past week I did plasmid digestion with XbaI (digested plasmid is looking good that time) and CIP it but didn't inactivated CIP. I think that as a consequence, my subsequent ligation step is poor and I've got clones that are only self-ligation. d(T_T)b

When I did the digestion again with the same condition, I have noticed that my plasmid DNA gets badly degraded after restriction digest. What's wrong with this?

I also did a "no enzyme digest" just add more water instead of enzyme with the same master mix to make sure there is no contamination in my reaction or self degradation of my DNA. These are my conditions:

Conditions: Digestion at 37C 30 min, 65C 20 min
1. Plasmid DNA digested with XbaI (Real digest)
2. Plasmid DNA digested with XbaI (Real digest with CIP)
3. Plasmid DNA digested with no enzyme but digested as I'd for a restriction digest

After digestion,
I took half of 3.(No-enzyme digest with CIP) and put into a new tube (4.No-enzyme digest)

I added CIP into 2. and 3. (60C 30 min)

and run a gel with 5 lanes of (below) and the results showed as follow:
1. real digest : smear
2. real digst with CIP : smear
3. no-enzyme digest with CIP : smear
4. no-enzyme digest : 3 normal bands
5. Undigested DNA : 3 normal bands

Is it means that my plasmid DNA degrade when I added only XbaI and also degrade with only CIP has been added ?

Why the same restriction condition wasn't work with a same batch of plasmid?

I made a few aliquots of these two enzymes, is it possible that both of these 2 aliquots contaminated with DNase? Do you know what else might be an obsticle? d(@_@)b


Sorry for too long story. Anyway, I'll be waiting for your advise.

Thanks in advance.

Bo d(^@^,)b

Did your "no enzyme digest" mixture have restriction enzyme buffers? The buffers contain magnesium, a required cofactor for DNAse activity. If your plasmid prep has DNAse contamination, but little or no magnesium (e.g. it is stored in TE) then addition of the buffer would be enough to see degradation. If your mock digestion did not have restriction enzyme buffers, you would not see degradation. If your mock digestion has RE buffer, then I have no easy explanation. If it does, then your DNA probably has DNAse contamination, and could be purified by phenol/chloroform extraction and ethanol precipitation. Are you preparing the DNA from an endA+ strain? If so, I'd recommend switching to an endA- strain.