Universe High-Fidelity Hot Start DNA Polymerase
Description
Universe High-Fidelity Hot Start DNA Polymerase is a superior
enzyme in this category for robust PCR with extreme fidelity,
featuring 53x error rate lower than Taq polymerase and extension
rate as fast as 1 sec / kb*. Universe High-Fidelity Hot Start DNA
polymerase possesses 5´→ 3´ polymerase activity, 3´ → 5´
exonuclease activity and will generate blunt-ended products. The
polymerase is also capable of amplifying long fragments such as
20kb genomic DNA and 40kb λDNA. Our suggested applications
include cloning, sequencing, genomic DNA amplification and high
throughput PCR, etc.
* The extension rate varies with individual application.
Components
Contents
Cat#:B21101 100U Cat#:B21102 250U Cat#:B21103 500U
(500 rxns)
(250 rxns)
(100 rxns)
Universe High-Fidelity
Hot Start DNA
polymerase (1 U /µl)
100 µL
250 µL
500 µL
2 x Universe buffer
1.25 mL x 2
1.25 mL x 5
1.25 mL x 10
dNTP Mix (10mM each)
10 x Loading buffer
25 µL Reaction 50 µL Reaction
Volume
Volume
Contents
100 µL
250 µL
500 µL
1.25 mL
1.25 mL x 3
1.25 mL x 5
Storage
• All reagents should be stored at -20 °C
Universe High-Fidelity Hot Start
DNA polymerase (1 U/µL)
1). All components should be mixed as recommended below prior to
use.
0.02 U/µLa
dNTP mix (10 mM each)
0.5 µL
1 µL
200 µM each
Variable
Variable
Variableb
Upstream primer (10 µM)
1 µL
2 µL
0.4 µM
Downstream primer (10 µM)
1 µL
2 µL
0.4 µM
2x Universe buffer
12.5 µL
25 µL
1x
Distilled water (dH2O)
To 25 µL
To 50 µL
-
Total reaction volume
25 µL
50 µL
-
a.The recommendation for final enzyme concentration is 1 U/ 50 µl,
but it can be varied in a range of 0.5 – 2 U / 50 µl, if needed.
b.General guidelines for low complexity DNA (e.g. plasmid, virus or
λDNA) are: 10 pg–30 ng per 50 µl reaction volume; for high
complexity genomic DNA, the amount of DNA template should be
50 – 400 ng per 50 µl reaction volume. cDNA synthesis reaction
mixture is used as template, the volume should not exceed 10 % of
the final PCR reaction volume.
Notes: Add the components into sterile PCR tubes while mixing
gently.
2).Place PCR tubes to a PCR cycler.
3).Perform PCR reaction using optimized cycling conditions.
Suggested cycling parameters for using Universe Hot Start DNA
Polymerase are provided below. Analyze PCR amplification
products on a 0.7–1.0% (w/v) agarose gel.
Segment
Number
3-Step Protocol
2-Step Protocol
Of Cycles Temperature °C Duration Temperature °C Duration
1
Notice
Protocol
1 µL
DNA template (100ng / µL)
1.Initial
Denaturation
1. All reagents have been optimized for use together and any
modifications or alternative uses are prohibited.
2. Before each step, check to make sure every reagent is fully liquid
and resuspended prior to use.
0.5 µL
Final
Concentration
2. PCR
25-35
b
95
3 min
(30 sec)a
95
3 min
(30 sec)a
95
15 sec
95
15 sec
55-65c
72
15 sec
15-30
sec/ kb
72d
15-30
sec/ kb
3. Final
Extension
1
72
5 min
72
5 min
4. Hold
1
4
∞
4
∞
a. This mix is based on a hot-start DNA polymerase, the
pre-denaturation activation condition should be set to
95°C for 3 minutes (for genomic DNA and cDNA) or 30sec (for
plasmid DNA and virus DNA) to thoroughly activate the enzyme.
b. Optimized cycling parameters may not necessarily be transferable
between thermal cyclers. Consult the instrument manufacturer’s
recommendations if further optimization of cycling parameters is
required.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]
c. The annealing temperature set up should be based on the Tm of
the primers.
d. For primers with annealing temperatures ≥ 72°C, a 2-step protocol
is recommended.
Trouble Shooting
Non-specific products - Low molecular
weight discrete bands
No product at all or low yield
• High quality or purified DNA templates
are preferred to enhance the success
of PCR.
• Repeat and make sure that there are
no pipetting errors.
• Use fresh high quality dNTPs.
• Do not use dNTP mix or primers that
contain dUTP or dITP.
• Sample concentration may be too low.
Use more template.
• Template DNA may be damaged. Use
carefully purified template and make
sure template is not fragmented.
• Increase extension time.
• Increase the number of cycles.
• Optimize annealing temperature.
• Optimize enzyme concentration.
• Optimize the denaturation time.
• Check the purity and concentration of
the primers.
• Check primer design.
• Increase annealing temperature.
• Decrease extension time.
• Decrease enzyme concentration.
• Titrate template amount.
• Decrease primer concentration
Non-specific products - High molecular
weight smears
• Decrease enzyme concentration.
• Decrease extension time.
• Reduce the total number of cycles.
• Increase annealing temperature or try
2-step protocol.
• Vary denaturation temperature.
• Decrease primer concentration.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]