Ghost plasmid of pBluescript

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed Mar 23 18:40:10 EST 1994


In the most recent issue of TIBS, I reviewed the discussion of an extra band
seen by some people when using Stratagene's pBluescript phagemid vectors:

@article{Hengen1994Martibs, 
author = "P. N. Hengen",
title = "Methods and Reagents: Ghost plasmid of pBluescript",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "3",
pages = "139-140",
month = "March",
year = "1994"}

Recently, I received an e-mail response from one reader with comments
he would like to share with the net. So, here it goes...

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Date: Thu, 17 Mar 94 11:05
From: QUIRKPG at VMS1.ACADEMIC-COMPUTING-SERVICE.BIRMINGHAM.AC.UK
To: PNH <PNH at NCIFCRF.GOV>

Dr Paul N Hengen,
NCI
Frederick, MD 21702-1201
pnh at ncifcrf.gov

Dear Dr Hengen,

I was interested to read your TIBS article about the ghost plasmid
of pBLUESCRIPT. Over the course of four years spent in Prof. Terry
Krulwich's lab, Mount Sinai School of Medicine, New York, we observed
similar "phantom plasmids" following transformation of various E. coli
strains (DH5-alpha MCR and F'lacIq, JM109) with ligation mixes using
pUC18 and pGEM series vectors. DH5-alpha colonies were white on 
IPTG/X-gal plates. They contained plasmids which were not cut 
by any of the MCS restriction enzymes. They were, however, cut by ScaI
and, if my memory is correct, AvaII. We concluded that they were 
spontaneous deletions from the vectors, losing the MCS and at least part 
of the lacZ fragment, but retaining ori and amp. ScaI cut them once, 
yielding linear fragments of varying sizes, but always smaller than intact
vector. As you suggest, Tn or IS factors may well have been involved;
our DH5-alpha MCR appears to contain at least one IS10 element (J. Bacteriol.
175, 8053).

I hope this information may be of help to you. Feel free to pass it on to 
any of your colleagues. 

Sincerely,


Philip G Quirk
School of Biochemistry
University of Birmingham,
Birmingham, B15 2TT, U.K.
QUIRKPG at VMS1.ACADEMIC_COMPUTING_SERVICE.BIRMINGHAM.AC.UK

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