Optimization of Parameters:

1. Template: The quality and quantity of the

Description:
The PerfectRead Plus (Pfr Plus) polymerase is an
upgraded version of proofreading DNA polymerase.
Compared with conventional DNA polymerase, Pfr
Plus was designed to carry out extreme long range
PCR reactions. When operating long range PCR, the
enzyme system must overcome several major
problems, such as the secondary structure of
template and misincorporation of dUTP. Ten Giga
Bio solved these problems in our Pfr Plus system by
blended PerfectRead polymerase with archaeal
dUTPase and specially optimized buffer system.
Therefore, Pfr Plus polymerase can easily amplify up
to 30 kb target DNA from lambda DNA or 20 kb from
human genomic DNA while maintaining
exceptionally high fidelity. In short, PerfectRead plus
system is your best choice to deal with long range
PCR.
DNA template is crucial for long-range PCR.
Always carefully prepare your template
DNA and the recommended quantities are
listed below:
DNA Standard PCR Long Range PCR Protocol
Protocol
Genomic 5 ng - 500 ng 100 ng 500 ng
DNA
Plasmid DNA 1 pg - 10 ng 100 pg 10 ng
cDNA 20 ng - 500 ng 200 ng 750 ng
2. Primer: Select oligonucleotide primers (20-
40 nt) with GC content of 40-60 % are
recommended Primers with higher melting
temperatures (Tm 62-70) are better, since
high annealing temperatures (65-70 ) is
preferred in long range PCR reactions.
3. Deoxynucleotides and additives: The
recommended final concentration of dNTPs
is 300 M each (higher than normal PCR).
Optional addition of PCR enhancing agents,
such as betaine or DMSO, may help to
reduce the secondary structures and
enhance PCR results.

Materials supplied:
1. PerfectRead plus DNA polymerases
(2 units/L) in storage buffer.
2. 5X PerfectRead plus buffer (1 mL/vial)
provides a final 2 mM Mg 2+ concentration
in the PCR reaction.
3. 5 M betaine (1mL/vial)

Applications:
1. Generation of large blund-ended DNA
products for cloning and expression.
2. Genomic mapping
3. Vector construction
4. High fidelity PCR
5. Site-directed mutagenesis
Reaction Mixture for Recommended PCR assay: Long Range PCR Protocol (10-20 kb):

Component Amount per reaction Number of Step Temperature Time

cycles
DNA template variable
10 M forward Primer 1 L 1 Initial 94oC 2
denaturation minutes
10 M reverse Primer 1 L
25-35 Denaturation 94oC 15-30
10 mM each dNTP 1 L
seconds
5X PerfectRead plus reaction 10 L
Annealing/ 68oC 30
buffer
Extension seconds/kb
PerfectRead plus DNA 0.5 L
1 Final 68oC 10-15
polymerase
Extension minutes
Nuclease-free water to 50 L
Hold 14oC --
Total volume 50 L

Note:
Note:
When using GC-rich templates or amplifying products 10
To prevent primer degradation or mispriming, we
kb fragments, 2-step PCR is recommended
recommended the reaction mix should keep on ice
Change the extension temperature to 68.
before PCR amplification started.
In order to obtain the best PCR results, it is
Long Range PCR Protocol with very long targets
important to optimize the reaction parameters,
(>20 kb):
such as additives or PCR cycling conditions.

Number of Step Temperature Time

Standard PCR Protocol (up to 10 kb):
cycles

1 Initial 94oC 1-2

Number of Step Temperature Time
denaturation minutes
cycles
20 Denaturation 94oC 15-30 seconds
1 Initial 94oC 2
denaturation minutes
Annealing/ 68oC 12 minutes
o
25-35 Denaturation 94 C 15-30 Extension
seconds
12-17 Denaturation 94oC 15-30 seconds
o
Annealing 45-72 C 15-30
seconds Annealing/ 68oC 12 min + 20

Extension 72oC 15-30 Extension sec per cycle

seconds/kb 1 Final 68oC 15 minutes

1 Final 72oC 10 minutes Extension

Extension Hold 14oC --

Hold 14oC --