Trouble with miniprep- DNA contamination

Paul N Hengen pnh at ncifcrf.gov
Thu Sep 26 12:44:55 EST 1996


David Konerding (dek at socrates.ucsf.edu) wrote:

> Hi, folks.  I've been working on doing a miniprep of overnight
> cultures for a few weeks and have been having some technical difficulties.
> Nobody in the lab is able to figure out why this is happening.
> 
> I've created a plasmid vector using ligation which I transform into
> DH5-alpha cells.  I grow the transformed cells on LB + ampicillin plates and
> innoculate a LB+AMP overnight culture using the colonies on the plates.
> 
> Taking a saturated 3mL culture, I follow a fairly standard miniprep
> protocol (from 'Short Protocols in Molecular Biology') and run the DNA out
> on a 1% agarose gel.  With a 1KB ladder lane and a control lane containing
> uncut vector from a previous experiment, I can see that I am purifying
> the plasmid DNA.
> 
> However, there is contamination.  My vector is ~2.7k and I see a strong
> band at approx. 12kb that is a brighter than my vector band.  We have
> puzzled over whether this could be 'denatured supercoiled' plasmid or possibly
> RNA or chromosomal DNA, but it seems resistant to specific restriction
> endonucleases.  Additionally, if I miniprep DH5-alpha cells without transforming,
> I still get the contamination band.  These cells do not have any other plasmid
> already in them.
> 
> Any ideas on how I can identify and eliminate this band?  As far as I can tell
> I am following the miniprep procedure (cell pellet into 100ul GTE, resuspend,
> add 200ul alkali lysis solution, on ice 5 min, add 150ul 3M NaOAc, on ice 5min,
> spin down, keep supernatant, do ethanol precip and raiise up DNA pellet in TE.)

I'm pretty sure I know what the band is, but I cannot explain why you get it
when you lyse the host cells without ANY plasmid, if that is what you did. You
say above that the cells don't have ANY OTHER plasmid. I take it that you are
getting the "ghost" band when isolating DNA from cells that contain plasmid
DNA. If that is true, you are probably experiencing the Freddy Kreuger plasmid
band as described by other netters. It is formed by the alkaline lysis
technique. To rid yourself of the band, i.) digest it away with T5 exonuclease
ii.) isolate the plasmid DNA using a non-alkaline-lysis technique such as
boiling, or iii.) cut the linear restricted DNA out from the gel for your next
experiment. A followup article on the ghost plasmid will be published in the
October 1996 issue of TIBS.

@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 = "mar",
year = "1994"}

@article{Sayers1996,
author = "J. R. Sayers
     and D. Evans
     and J. B. Thomson",
title = "Identification and eradication of a denatured
{DNA} isolated during alkaline lysis-based plasmid
purification procedures",
journal = "Anal. Biochem.",
volume = "241",
number = "2",
pages = "186-189",
month = "oct",
comment = "ghost plasmid of pBluescript",
year = "1996"}

@article{Hengen1996Octtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Eliminating ghost bands from plasmid preps",
journal = "Trends in Biochemical Sciences",
volume = "21",
number = "10",
pages = "xxx-xxx",
month = "oct",
year = "1996"}

--
*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
* - - -  Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - -  *
* -  TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - -  *
* - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/  *
*******************************************************************************



More information about the Methods mailing list