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Epicentre Forum 3 (3)
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Yutaka Tagaya Metabolism Branch, National Cancer Institute, National
Institutes of Health, Bethesda, MD 20892
T4 DNA ligase reactions are widely used in molecular biology for subcloning,
ligation-mediated PCR (LM-PCR) and DNA library construction. Conventional
ligation requires a long incubation period (typically overnight) at temperatures
ranging from 12-22°C. Using Epicentre's Fast-Link™ DNA Ligation
and Screening Kit,* we report that ligation reactions containing blunt-end
DNA or PCR products can be performed in 15 minutes at room temperature
with higher efficiency than conventional ligation. This system would
be useful for applications where high-efficiency DNA ligation is required.
Comparison of Fast-Link rapid ligation and conventional ligation
with blunt-end, double-stranded DNA
To compare Fast-Link ligation with conventional ligation, 10 µg
of pBluescript® SK (Stratagene) were digested with EcoR V
to generate blunt-ended, linearized plasmid that was then treated with
calf intestinal alkaline phosphatase (Boehringer-Mannheim) to prevent
self-ligation. Blunt-ended PCR fragments were generated using Vent® DNA
polymerase (New England Biolabs) with 30 cycles of amplification. The
amplified fragment (350 bp) was subjected to gel electrophoresis in a
1.2% agarose gel and the gel fragment containing the amplified DNA was
excised. Extraction of the DNA was performed using the Geneclean® kit
(BIO 101) according to the manufacturer's instructions. For conventional
ligation, 50 ng of the insert were incubated with 150 ng EcoR
V-treated plasmid, 4U of T4 DNA ligase purchased from Stratagene, standard
buffer and 1 mM ATP in a total volume of 15 µl. The reaction was
incubated at 22°C for 12 hours. Ligation using the Fast-Link Kit
was performed according to the manufacturer's instructions. The ligation
reaction contained 50 ng of insert DNA, 150 ng of digested plasmid, 2U
of Fast-Link T4 DNA Ligase, and 0.5 mM ATP in a total volume of 15 µl
in 1X Fast-Link Ligation Buffer. The reaction was incubated for 15 minutes
at room temperature, and then at 70°C for 15 minutes. 2.5 µl
of the ligation mixture from each reaction were used to transform XL2-Blue
competent cells (Stratagene), and one-third of the total transformed
cells were plated on LB Amp/IPTG/X-gal plates.
Table 1 shows the ligation efficiency calculated from the colony number
obtained after a 12-hour incubation of the plates at 37°C. Ligation
with the Fast-Link Kit was almost 10-fold more efficient than conventional
ligation.
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Conventional
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72%
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0.7 x 105
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Fast-Link
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68%
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7.6 x 105
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Comparison of Fast-Link rapid ligation and conventional ligation
with a TA cloning vector
A similar experiment was designed to compare the two ligation techniques
with the TA cloning system. PCR product was generated using Taq DNA polymerase.
After isolation and quantitation of the PCR fragment as described above,
the fragment was ligated to the pT7Blue vector (Novagen). Briefly, 25
ng of the PCR product (500bp) were ligated to 50 ng of the vector (2.8
kb)
(vector to insert ratio of 1:3) in the same reaction conditions as described
above. The conventional ligation was incubated at 16°C overnight.
The Fast-Link ligations were incubated either at room temperature for
15 minutes or 16°C for 1 hour. 2.5 µl of each 15 µl ligation
reaction were used to transform XL2-Blue competent cells. One-fifth of
the transformed cells were plated onto LB Amp/IPTG/X-gal plates. As shown
in Table 2, the Fast-Link Kit yielded almost a 20-fold increase in the
ligation efficiency over the conventional method. In addition, incubation
at 16°C for one hour resulted in higher ligation efficiency than
the 15-minute, room-temperature incubation.
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Conventional
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16°C / overnight
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50%
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0.12 x 104
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Fast-Link
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16°C / 1 hr
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27%
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4.3 x 104
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Fast-Link
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Room temp. / 15 min
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44%
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1.8 x 104
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To assess the recombination efficiency, white colonies from each plate
were picked and plasmid DNA was purified using the Wizard™ mini-prep
kit (Promega). The isolated plasmid was digested with Hind III
and BamH I in a 50 µl volume for 1 hour at 37°C and
then subjected to gel electrophoresis in a 1.2% agarose gel in 1X TAE
buffer. Figure 1 indicates that the Fast-Link Kit and the conventional
method showed almost comparable efficiency of recombinant clones (over
60% of recombinants). Collectively, these results indicate that Fast-Link
ligation resulted in 5- to 20-fold higher ligation efficiency than conventional
ligation with a similar recombination efficiency.
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Figure 1. Results of recombinant screening. Five colonies
were picked from plates generated with either conventional ligation
or the Fast-Link Kit. Plasmid DNA isolated using the Wizard mini-prep
kit was digested with Hind III and BamH I to isolate
the insert (500 bp). The marker is a 1 kb ladder (Life Technologies). |
The Fast-Link DNA Ligation and Screening Kit provides an alternative
ligation method that is faster and more efficient than conventional ligation
with T4 DNA ligase. This system is useful for applications requiring
high ligation efficiency.
Fast-Link™ DNA Ligation and Screening
Kit
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