lambda digest: the pen-ultimate solution ?

suter at VAX.MPIZ-KOELN.MPG.dbp.de suter at VAX.MPIZ-KOELN.MPG.dbp.de
Thu Sep 10 12:32:46 EST 1992


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
----------------------------------> cut here <----------------------------------
Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10
5000 Koeln 30
Tel. xx49-221-5062.221           fax. xx49-221-5062.21
suter at vax.mpiz-koeln.mpg.dbp.de
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