Restriction enzyme digestion troubles
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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.
Newsgroup archive
> 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 Fair of 5 votes.
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what kind of RE? what is source of DNA? plasmid or chromosomal?
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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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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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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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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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I am gettting very faint bands after digestion with restriction enzymes. How to solve this problem
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Related resource
Double restriction digestion of DNA
Rsa digestion.
Labelling of oligos: an interesting phenomenon
Non-radioactive labelling of DNA
High temp. restriction enzyme digests
RANDOM primer labelling efficiency
REBASER: The Restriction Enzyme Database
Integrated Enzyme Database (IntEnz)
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