Genomic Digest Difficulties
broe at aardvark.ucs.uoknor.edu
Tue Jul 20 07:21:00 EST 1993
In article <CAFE13.CIB at ncifcrf.gov>, pnh at fcsparc6.ncifcrf.gov (Paul N Hengen) writes...
>In article <1352 at grivel.une.edu.au> iwatson at metz.une.edu.au (IAN WATSON) writes:
>> .....I acheived a good yield, however, whenever I try to
>> digest them (with EcoRI and HindIII) They do not digest at all. Initially I
>> thought that there may be proteins present in the prep or perhaps phenol which
>> was interfering with the digest. So I carried out a phenol extraction again
>> and ethanol precipitated them again. After this the digest failed again. So I
>> thought that there was an outside chance that the TE buffer that the genomic DNA
>> was dissolved in had too high a concentration of EDTA causing the nucleases to
>> fail so I ethanol precipitated them again and then dissolved in new TE. However,
>> This also failed.
>I would like to suggest you don't use TE buffer for your final DNA sample.
>I ran into problems of this sort when first isolating plasmid DNA from
>mini-preps. The cleanest digests were obtained when the DNA pellet was washed
>several times with 70 % ETOH (breaking up the pellet with a pipet tip while
>pumping the ethanol/DNA solution). The DNA is spun down afterward and this is
>repeated at least 3 times with fresh ETOH. The final pellet is dried and
>resuspended in sterile distilled water.
>Paul N. Hengen
>National Cancer Institute
>Frederick Cancer Research and Development Center
>Frederick, Maryland 21702-1201 USA
My 2 cents worth are:
1) If the genomic DNA isolation was done in parallel with another sample
that was digested then it must be something that is DNA related.
2) Was a parallel control digestion done using, for example, a pUC template
or some other plasmid to test the enzymes/reaction buffers/etc? If not,
then the enzymes could have gone off.
3) Sorry Paul but I don't agree with you. Replacing TE with just water is
not necessarly a good idea. The purpose of TE is two fold, one to provide
a known pH and the other to have a trace amount of EDTA there to chelate
any heavy metals that may be required for nuclease activity. However,
since the concentration of Tris and EDTA in TE is so low, when the DNA
dissolved in TE is added to the restriction digestion reaction, the
reaction buffer and magnesium present there more than exceeds the chelating
capacity of the trace amount of EDTA in the TE used to dissolve the DNA.
Sure it works to dissolve the DNA in just water, but I prefer TE as a
safety measure in case there is any chance of nucleases around.
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