lambda digest: the pen-ultimate solution ?

Martin Kennedy cytogen at chmeds.ac.nz
Sat Sep 12 04:51:14 EST 1992


In article <1992Sep11.131419.25990 at gserv1.dl.ac.uk>, suter at VAX.MPIZ-KOELN.MPG.dbp.de writes:
> Mike Grosse wrote:
> 
> <Subj:	lambda won't cut
> <
> <Hello netters,
> <
> <A colleague provided us with some lambda clone DNAs "purified" from plate
> <lysates.  They won't cut with EcoR1.  We tried further purification with
> <additional phenol/chloroform extractions.  The uncut DNA looks good on a
> <gel, and the OD's are good.  The inserts should cut out with EcoR1 based on
> <the way the library was made (R1 linkers).  We haven't worked with lambda
> <in quite a while, but I imagine that some of you out there are cutting
> <yours successfully as you read this.
> <
> <What do you suggest?
> <
> <                                Mike Grosse
> <                                McGraw lab
> <                                University of Georgia
> 
> i had EXACTLY the same problem two months ago: same enzyme, same technique,
> same results ! my boss suggested that there is something in the LB/top agarose
> that inhibits digestion, and you won't be able to get rid of it with 
> standard procedures (heavy metal ? polysaccharides ? i don't know). anyway,
> these same compounds may trouble you when you ligate in low melting agarose
> also...
> 
> after many different purifications, all in vain, i purified the DNA
> over a QIAGEN column, and presto: perfect DNA, perfect Eco digest, perfect
> ligation.
> 
> if you don't have these kind of columns, perhaps you should use another
> kind that is used for DNA preparation in your lab, i just followed
> the dna miniprep protocol (perhaps sephadex would also work, altho the principal
> is completely different).
> 
> good luck !
> 
> clemens
> 

A few years back I read a paper (sorry - can't remember where, but possibly
Gene Analysis Techniques)  where someone tried numerous procedures to remove
the unidentified nasty(s) which inhibit cutting of lambda preps.  They claimed
that passing the lambda DNA through a Sepharose 4B (or 6B - again, my memory
fails me) column usually solved the problem. I haven't tried it.  Why not
transform the DNA back into E.coli  (works at low frequency) and make a new
prep (I always find new preps more likely to work than trying to get old ones
to cut)?  The other desparate measure I've used is multiple phenol/CHCl3
extractions (ie 5 or 6), which occasionally works.

Cheers,

Martin



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