Download HotStart DNA Polymerase

HotStart Storage Buffer
The enzyme is supplied in 20 mM Tris-HCl pH 8.5, 100
mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20,
0.5% NP40, 50% glycerol.
Suggested Protocol
This protocol serves as a guideline for primer extensions. Optimal reaction conditions such as incubation
times, temperatures, and amount of template DNA may
vary and must be individually determined.
HotStart DNA Polymerase
Conc.: 5 units/µL
10X HotStart Buffer
(15 mM MgCl2)
1.5 mL
Notes:
Cat. No.
M016TP30
Size
(Units)
250
25 mM
MgCl2
1.5 mL
M016TP40
500
1.5 mL
1.5 mL
M016TP70
2500
5 x 1.5 mL
5 x 1.5 mL
Store at -20°°C. FOR RESEARCH USE ONLY
General Description
HotStart DNA Polymerase is a thermostable DNA
Polymerase that is activated by heat treatment. It is
chemically modified to remain inactive until time,
temperature and pH conditions are optimal. This results
in higher specificity and greater yields when compared to
standard DNA polymerases.
o
HotStart requires a 15-minute activation step at 95 C,
activating the enzyme at a temperature well above
optimal annealing. During hot start, primers bind only to
their specific target, and polymerase activity is directed
exclusively to that target. Only the region of interest is
amplified, which increases sensitivity and yield while
reducing non-specific background amplification.
Since the enzyme is chemically modified for hot start (no
antibody), there is no material from animal sources, i.e.,
no possibility for biological contamination
Key Features
Automated HotStart enzyme for increased
specificity and product yield
Successful multiplex reactions saves time and
reagents
Designed to diminish the formation of non-specific
product
Detection of low target copy number
Unit Definition
One unit is defined as the amount that incorporates
10 nmoles of dNTPs into acid-precipitable form in
30 minutes at 72°C under standard assay conditions.
10X HotStart Reaction Buffer
Combination of KCl and (NH4)2S04,15 mM MgCl2,
1% Tween 20.
M016TP30- July 2007
HotStart DNA Polymerase requires an activation
step of 15 minutes at 95°C.
Set up reaction mixtures in an area separate from
that used for DNA preparation or product analysis.
1. Thaw 10X HotStart Buffer, dNTP mix, primer
solutions. It is important to mix the solutions
completely before use to avoid localized
concentrations of salts.
2. Prepare a master mix according to Table 1. The
master mix typically contains all the components
needed for extension except the template DNA.
In some applications, more than 1.5 mM MgCl2, as
provided in the 1X HotStart Buffer, is needed for
optimal results. For this reason, 25 mM MgCl2 is
included in the kit. Table 2 provides the volume of
25 mM MgCl2 to add to the master mix if a higher
MgCl2 concentration is required.
Table 1. Reaction components
(master mix and template DNA)
Component
10X HotStart Buffer
dNTP mix
(12.5 mM of each)
Primer A
Primer B
HotStart DNA
Polymerase
Distilled water
Template DNA
TOTAL volume
Vol./reaction
5 µL
Variable
Variable
Final Conc.
1X
0.2 mM of
each dNTP
0.1 to 0.5 µM
0.1 to 0.5 µM
1 µL
5 units
Variable
Variable
50 µL
---Variable
----
0.8 µL
Table 2. MgCl2 concentration
Final MgCl2 conc.
in reaction (mM)
Additional volume
of 25 mM MgCl2
per reaction (µL):
Related Products
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0
1
2
3
4
5
6
3. Mix the master mix thoroughly and dispense
appropriate volumes into reaction tubes. Mix gently,
e.g., by pipetting the master mix up and down a few
times.
4. Add template DNA to the individual tubes containing
the master mix.
5. Program the thermal cycler according to the
manufacturer´s instructions. Each program must
start with an initial heat activation step at 95°C
for 15 minutes.
For maximum yield and specificity, temperatures
and cycling times should be optimized for each new
template target or primer pair.
6. Place the tubes in the thermal cycler. A typical
thermal cycling program is shown below:
95°C for 15 min.
Activate polymerase
& denature template
Description
Taq DNA Polymerase
with 10X Standard Reaction Buffer
Taq DNA Polymerase
with 10X Ammonium Reaction Buffer
Taq DNA Polymerase
with 10X Combination Reaction Buffer
Taq DNA Polymerase
with 10X Mg Free Standard Buffer
Taq DNA Polymerase
with 10X Mg Free Ammonium Buffer
Taq Complete 1.1X Master Mix
HiFed Polymerase
Long Range PCR Enzyme Blend
Long Range Complete 1.1X Master
Mix
TM
RedPOL DNA Polymerase
TM
RedMix Plus 2X Master Mix
Buffer Optimization Kit
Enhancer Optimization Kit
dNTP Mix, 50 mM
(12.5 mM of each dA, dC, dG and dT)
Cat. No.
M001TP20
M005TP20
M008TP40
M004TP20
M007TP40
M010TP20
M018TP30
M020TP20
M021TP20
M013TP40
M014TP20
M061RB33
M062RB49
M066DM80
NOTICE
In certain countries, patents cover the PCR process. This
product is intended for researchers having a license to
perform PCR or those not required to obtain a license.
30 to 40 cycles:
95°C
30 sec.
Denature template
45 to 65°C 30 sec.
Anneal primer
72°C
Elongation*
1 to 5 min.
72°C for 5 min.
Final Elongation
*Recommended time is 1 min. per kb of target
Quality Control
Endonuclease, exonuclease and priming activities are
not detected after 3 hours incubation of 1 µg of pUC19
plasmid DNA and 0.5 µg EcoR I digested lambda
phage DNA at 72°C in the presence of 40 units of
HotStart DNA Polymerase.
Tween 20 is a registered trademark of ICI Americas, Inc.
M016TP30- July 2007
2222 Michelson Drive
Suite 615
Irvine, CA 92612
T: +1-949-226-8094
F: +1-949-608-1975
www.alliancebio.com
For Technical service:
[email protected]
FOR RESEARCH USE ONLY