GHOST-BAND UN-MASKED

Helge Weissig helgew at LJCRF.EDU
Thu Apr 14 12:52:46 EST 1994


At 03:33 PM 04/14/94 +0100, BSS032 at BANGOR.AC.UK wrote:
>Dear Ghost-Band-Busters,
>
>A few weeks ago I posted an offer of an enzyme which degrades ssDNA, linear
>dsDNA and nicked circles BUT DOES NOT DAMAGE native plasmids (cccDNA or RFI).
>
>NOW for the result. I took 30 ul of buffer (glycine at pH9/KOH, 50 mM, MgCl2
>at 10 mM) containing approx. 3 ug of pUC19 (QIAGEN prepped) containing the
>famous 'Ghost-Band' (supplied by Dr. Jimmy Thomson, Max-Planck-Inst,Goettingen
>Germany). this was split into 3 aliquots and treated with either nothing,
>1 ug T5 exonuclease, or 0.01 ug T5 exo. All three were incubated at 37degC
>for 20 mins. Then heat killed at 70degC (ortherwise the DNA/protein complex
>is gel shifted), then run agarose.
>
>
>RESULT. The untretaed plasmid prep. contained several bands typical of
>QIAGEN prepped DNA i.e. cccDNA, some nicked (relaxed or RFII), some genomic
>DNA (running high up the gel) and our 'GHOST" running slightly ahead of the
>brightest (native plasmid) band. HOWEVER, the T5 exo treated samples had
>both lost their 'GHOSTS', genomic and nicked bands, but retained the strong
>ccc (or RFI) plasmid band!!!
>
>We are doing a couple of more experiments to tie this up, BUT we can already
>say that the 'GHOST' has nothing to do with phage origins of replication as
>some netters suggested.  It seems likely that the band issome form of replicat-
>ive intermediate singlestranded DNA. We are checking now but strongly suspect
>that it IS circular single-stranded plasmid. Hence it is resistant to restriction enzyme cleavage etc.
>
>Jon R. Sayers *   AND Jimmy Thomson
>
>Jon R Sayers (Cell and Molecular Biology), Biochemistry Dept.,
>University College North Wales, Univ of Wales, Bangor, LL57 2UW.
>Tel +44 248 38 23 54
>Fax +44 248 370731
>Email bss032 at uk.ac.bangor


Hi netters,

        I was following this threat with quite some interest, wondering, whether it would eventually help explain my 'ghost bands'... unfortunately it did not.

The problem I have is a predominant mini-prep (handmade, no kits, no silica) product from Bluescript transformations. It is really the only band I get sometimes and in its amount comparable to 'normal' plasmid.
Its size is somewhere around 1.5 to 1.8 kb. It is cutable with a few enzymes (at least appears to be cut on EtBr gels) but usually contains no insert.

Ok, the easiest way would be to sequence it (or try to) but maybe someone already did.

I strongly doubt it is ssDNA, since the intensity of EtBr staining and the sharpness of the band on an agarose gel are very similar to dsDNA.

any other ghost busting suggestions?

thanx,
Helge



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Helge WEISSIG                              e-mail: helgew at ljcrf.edu
La Jolla Cancer Research Foundation        phone: (619) 455 6480 x253
10901 N. Torrey Pines Rd.                  FAX: (619) 455 0181
La Jolla, CA 92037
U.S.A.

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