Genomic Digest Difficulties

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Mon Jul 19 13:58:15 EST 1993


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



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