Download Tech Notes Use of Plasmid-Safe™ to Prevent Cloning Artifacts Due

Tech Notes
Use of Plasmid-Safe™ to Prevent Cloning Artifacts Due to Bacterial
Chromosomal DNA
Ribozymes are catalytic RNAs that Even
following purification in CsCl/ethidium
bromide gradients, plasmid and cosmid
preparations may still contain contaminating bacterial chromosomal DNA. This contaminating DNA can be cloned along with
a target DNA resulting in cloning artifacts,
especially for low-copy number plasmids or
cosmids. Epicentre’s Plasmid-Safe ATP-Dependent DNase provides a novel method
for selectively removing contaminating
linear chromosomal DNA from plasmid
and cosmid preparations before their use
in cloning experiments. The Plasmid-Safe
protocol is shown below.
Plasmid-Safe Protocol
1. Isolate DNA from bacteria using standard mini-or maxi-prep protocols.
2. Resuspend the pelleted DNA in 1X
Plasmid-Safe Buffer and add ATP to a
final concentration of 1 mM ATP.
3. Add Plasmid-Safe to the reaction: 10
U for a mini-prep or 100 U for a 500 ml
prep.
4. Incubate at 37°C: 15 min for a mini-prep
or 2 hr for a 500 ml prep.
5. Inactivate Plasmid-Safe by incubation at
70°C for 15 min.
Note: If desired, the treated DNA can be
purified by ethanol precipitation, spincolumns, or organic extraction before further
manipulations.
Experiment 1. Elimination of Linear DNA
Resulting in KanR Colonies A mixture was
prepared containing equimolar amounts of
both a linearized (KanR) DNA and a supercoiled
(AmpR) plasmid. One aliquot of the plasmid
DNA mixture was treated with Plasmid-Safe;
a second aliquot was not treated and served
as the Control. The resulting plasmid samples
were then treated with T4 DNA Ligase, and the
ligation mixtures used to transform competent
cells. Transformed bacteria were plated onto
selective media. The Control sample gave rise to
404 KanR colonies (generated from the ligation
of linear DNA). In contrast, the sample treated
with Plasmid-Safe gave rise to only a single KanR
colony. Both samples gave rise to 500-700 AmpR
colonies when plated onto the appropriate
media, indicating no degradation of supercoiled
DNA with Plasmid-Safe. Thus, treatment with
Plasmid-Safe resulted in a 400-fold reduction in
unwanted linear DNA, while leaving supercoiled
DNA intact.
The following two experiments clearly
demonstrate the effectiveness of using
Plasmid-Safe to reduce cloning artifacts
due to contaminating chromosomal DNA.
Experiment 2. Elimination of Linear DNA Resulting in White Colonies Three µg of EcoR I-digested
bacterial genomic DNA was added to 2 µg of supercoiled pBS SK+ (Stratagene), a plasmid that
can be used for “Blue-White” screening assays on the appropriate media. Half of the DNA mixture
was treated with Plasmid-Safe; the other half was not treated and served as the Control. After heat
inactivation of the Plasmid-Safe enzyme, the DNA was digested with EcoR I, ligated overnight with T4
DNA Ligase (Epicentre) and transformed into Nova Blue™ competent cells (Novagen). The transformants
were plated on IPTG/X-gal-containing media. Blue colonies indicate reclosure of the plasmid; white
colonies indicate that bacterial DNA was inserted into the plasmid. As seen on plate A at upper right,
greater than 50%, of the colonies transformed by the control DNA sample were white colonies. In
contrast, only 1-3% of the colonies transformed by the Plasmid-Safe-treated DNA were white, (plate B
at upper right). This is equivalent to the assay background (white colonies seen when the plasmid is
restricted and ligated in the absence of any chromosomal DNA contamination). Thus, use of PlasmidSafe resulted in elimination of almost all of the linear DNA.
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