Bst 2.0 MS_Euro_mark.. - Lab-JOT

DNA Amplification & PCR
Bst 2.0 DNA Polymerase
Bst 2.0 WarmStart DNA Polymerase
DNA Cloning
DNA Amplification & PCR
Epigenetics
RNA Analysis
Sample Prep for Next Gen Sequencing
Protein Expression & Analysis
Cellular Analysis
Bst 2.0 – The Next Generation of Isothermal Polymerases
Bst 2.0 DNA Polymerase is an in silico designed homologue of Bacillus stearothermophilus DNA
Polymerase I, Large Fragment (Bst DNA Polymerase, Large Fragment). Bst 2.0 DNA Polymerase
posesses 5´→3´ DNA polymerase activity and strong strand-displacement activity but lacks 5´→3´
exonuclease activity. Bst 2.0 DNA Polymerase displays improved amplification speed, yield, salt
tolerance and thermostability compared to wild-type Bst DNA Polymerase, Large Fragment.
Figure 1: Bst 2.0 DNA Polymerase improves amplification speed
Yield (Abs.)
Strand Displacement Amplification (SDA) of BRCA1 from HeLa genomic DNA under optimal SDA conditions for each enzyme.
Data shows that the reaction reaches threshold faster with Bst 2.0 than with wild-type Bst DNA Polymerase.
Figure 2: Bst 2.0 DNA Polymerase has a greater range of
salt tolerance
Time to Threshold (min)
0.2
0.1
0.0
35
30
25
20
15
10
0 10 20
Time (min)
Salt Tolerance
wt Bst
Bst 2.0
Human Amplicon
wt Bst
Bst 2.0
Advantages:
• Fast polymerization
• Robust reactions with a broad range of
conditions and primer sets
• Flexible reaction conditions, including
a higher salt tolerance than Bst DNA
Polymerase
• Optimal reaction performance from
60-72°C
• Minimal effect of substitution of dTTP
with dUTP
• Highly pure product with minimal
lot-to-lot variation
• WarmStart feature enables room
temperature reaction set-up and
prevents non-templated addition
of nucleotides, increasing reaction
efficiency
Applications:
• Isothermal DNA amplification
• Applications requiring stranddisplacement
DNA synthesis
• DNA sequencing through high GC
regions
• Rapid sequencing from nanogram
amounts of DNA template
5
0 20 40 60 80 100 120 140 160
KCl (mM)
SDA amplification of BRCA1 from HeLa genomic DNA under optimal SDA temperature conditions
for each enzyme, with varying salt concentrations. Data shows that Bst 2.0 performance is minimally
affected by salt concentration.
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DNA Amplification & PCR
Bst 2.0 WarmStart DNA Polymerase
The WarmStart feature of Bst 2.0 DNA Polymerase is unique among isothermal polymerases. Like
“Hot Start” PCR polymerases, this feature prevents activity at temperatures below the optimal reaction
temperature. This enables room temperature reaction set up and minimizes undesirable reaction
products, thereby increasing reaction efficiencies.
In contrast to chemical modifications or antibodies commonly used with hot start PCR polymerases,
NEB’s Bst 2.0 WarmStart DNA Polymerase utilizes aptamer technology. Aptamers are
extensively modified, unique oligonucleotides which bind to the polymerase through non-covalent
interactions, inhibiting activity at non-permissive temperatures (< 50°C). Additionally, no separate
activation step is required for Bst 2.0 WarmStart DNA Polymerase.
While standard formulations of Bst DNA polymerase and homologs add non-templated nucleotides
to substrate during room temperature reaction setup, this undesirable activity is eliminated in this
WarmStart version.
Figure 3: WarmStart feature enables room temperature setup
Time to Threshold (min)
35
30
25
20
15
Bst DNA Polymerase
no pre-incubation
Bst DNA Polymerase
2 hrs 25°C
Time to Threshold (min)
5.5
5.4
5.3
5.2
5.1
5.0
4.9
Bst 2.0
no pre-incubation
Bst 2.0
2 hrs@25°C
Time to Threshold (min)
6.8
6.4
6.0
5.6
Bst 2.0 WarmStart
no pre-incubation
Bst 2.0 WarmStart
2 hrs@25°C
10
2 3 4 5
log (copies HeLa DNA)
4.8
4.7
2 3 4 5
log (copies HeLa DNA)
5.2
2 3 4 5
log (copies HeLa DNA)
For Bst DNA Polymerase and Bst 2.0, incubation of the sample at room temperature before beginning the SDA assay results in variable performance. For Bst 2.0 WarmStart, extended pre-assay incubation at room temperature
does not affect performance. The different scales highlight the increased reaction speed with Bst 2.0 and Bst 2.0 WarmStart DNA Polymerases over wild-type Bst DNA Polymerase.
Figure 4: Consistency of purity between lots of Bst 2.0
µg protein 20 4 2 1 0.5 0.25 0.13 0.063 0.031 M µg protein 20 4 2 1 0.5 0.25 0.13 0.063 0.031 M µg protein 20 4 2 1 0.5 0.25 0.13 0.063 0.031 M
Amount of protein loaded for 3 different lots is indicated in the above gels, Marker M is the Prestained Protein Marker (NEB #P7708).
Ordering Information
Product NEB # SIZE
Bst 2.0 DNA Polymerase M0537S/L/M 1,600/8,000/8,000 units
Bst 2.0 WarmStart DNA Polymerase M0538S/L/M 1,600/8,000/8,000 units
companion product
Isothermal Amplification Buffer Pack B0537S 6.0 ml
WARMSTART is a trademark of New England Biolabs, Inc.
New England Biolabs (UK) Ltd.
www.neb.uk.com
New England Biolabs France
www.neb-online.fr
www.neb.com
New England Biolabs GmbH, Germany
www.neb-online.de
For a complete list of international offices and distributors, please visit www.neb.com
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