|
Epicentre Forum 1 (2)
||
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 in Table 1.
Table 1. Plasmid-Safe Protocol
- Isolate DNA from bacteria using standard mini-or maxi-prep
protocols.
- Resuspend the pelleted DNA in 1X Plasmid-Safe Buffer and add
ATP to a final concentration of 1 mM ATP.
- Add Plasmid-Safe to the reaction: 10 U for a mini-prep or 100
U for a 500 ml prep.
- Incubate at 37°C: 15 min for a mini-prep or 2 hr for a
500 ml prep.
- Inactivate Plasmid-Safe by incubation at 70°C for 15 min.
Note: If desired, the treated DNA can be purified by ethanol precipitation,
spin-columns, or organic extraction before further manipulations. |
The following two experiments clearly demonstrate the
effectiveness of using Plasmid-Safe to reduce cloning artifacts due to
contaminating chromosomal DNA.
 |
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. |
 |
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 Plasmid-Safe resulted in elimination of almost all of the
linear DNA. |
|
