Technique / Molecular Biology / Enzymatic treatment of DNA/RNA


Restriction enzyme digestion troubles



Restriction enzyme digestion troubles

I start out with high molecular weight DNA. When I try and carryout my digestion it seems as though my DNA is being degraded by the enzyme and I have tried two different enzymes. When I just add water or buffer or water/buffer to the DNA it is fine i.e. high molecular weight. what should I do!!!!!!
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> I start out with high molecular weight DNA.


What kind? Plasmid? Chromosomal? What source? What purification method?



When I try and carryout my d=
> igestion it seems as though my DNA is being degraded by the enzyme


What else do you expect? It's a restriction enzyme. If you digest
chromosomal DNA you will see a smear.




and I =
> have tried two different enzymes. When I just add water or buffer or
wat=
> er/buffer to the DNA it is fine i.e. high molecular weight. what
should =
> I do!!!!!!


Test the enzyme on a standard template. However, most likely nothing is
wrong with your experiment. More likely, your interpretation needs some
refinement.
Regards,
Helen.
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> I start out with high molecular weight DNA.


Source?



>?When I try and
> carryout my digestion it seems as though my DNA is being degraded
> by the enzyme and I have tried two different enzymes.?


If the DNA is say human genomic then you won't see discrete bands on a
normal minigel and may not even resolve discrete bands on a larger gel.


For a nuclease test control, try an incubation in the RE buffer but
without the RE. If that is OK then you can rule out nucleases in your
water, DNA and RE buffer.


You can also try adding 1ug of lambda DNA to your genomic digest. If you
get clean discrete digested lambda bands of the correct size with the
genomic 'smear' in the background then your RE is fine and there is
probably nothing to worry about.
Duncan
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Last update 18-Apr-2004, Rating of 6 votes.

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By rama on 06-Dec-2008
iam using a low copy number palsmid it was isolated ina corect manner the conc is good but thereis digestion problem iam using cla1 enzyme even though the conc is good then why the digestion is not occuring
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By sdinesh on 03-May-2008
what kind of RE? what is source of DNA? plasmid or chromosomal?
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By GEETHA on 25-Oct-2007
when i do the digestion with smaller amount of DNA its working ,but same thing when i repeated for large amounts its not working,what is the problem?
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By Asad on 03-Aug-2007
I am doing SSAPs with Sea buckthorn Genomic DNA. Digestion problems, no smear in 1% agarose gel. Changed the concentrations many times. What I do and what could be the problems?
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By teshee on 02-Jan-2007
I am also facing the same problem, although the bands are visible, they are very faint to be photographed, there is very good resolution also
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By Kumara Swamy G.K. on 15-Jun-2006
When the DNA is digested, it is going to divide the total concentration of DNA into different fragments with different concentration. Hence it is obvious that the band will faint compare to the intact DNA. So use higher concentration of DNA for digestion
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By farzana on 18-Apr-2004
I am gettting very faint bands after digestion with restriction enzymes. How to solve this problem
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