5. Notes about reaction components
5.1. Enzyme
Phire Hot Start II DNA Polymerase possesses the
following activities: 5´→3´ DNA polymerase activity and a
weak 3´→5´ exonuclease activity. Phire Hot Start II DNA
Polymerase produces blunt ends and therefore blunt end
cloning is recommended. If TA cloning is required, it can
be performed by adding A overhangs to the blunt PCR
product with Taq DNA Polymerase, for example (protocol
available at www.thermoscientific.com/pcrcloning).
5.2. Phire Plant PCR Buffer
The 2X Phire Plant PCR Buffer has been optimized for
direct PCR from plant material. It contains the dNTPs and
provides 1.5 mM MgCl
2
concentration in the final
reaction.
5.3. Dilution Buffer
The Dilution Buffer has been optimized to release DNA
from a wide variety of different sample materials such as
plant leaves and seeds. This buffer is also suitable for
storing the DNA sample for 8 weeks at 4 °C. For long
term storage, it is recommended to transfer the
supernatant into a new tube and store at −20 °C. The
Dilution Buffer is sufficient for 500 dilution reactions
20 µL each.
5.4. Primers
The recommendation for final primer concentration is
0.5 µM. The results from primer Tm calculations can vary
significantly depending on the method used. Always use
the Tm calculator and instructions at
www.thermoscientific.com/pcrwebtools to determine the
Tm values of primers and optimal annealing temperature.
6. Notes about cycling conditions
6.1. Initial denaturation
In Direct PCR protocols, the initial denaturation step is
extended to 5 minutes to allow the lysis of cells, making
genomic DNA available for PCR.
6.2. Primer annealing
Note that the optimal annealing temperature for Phire Hot
Start II DNA Polymerase may differ significantly from that of
Taq-based polymerases. Always use the Tm calculator and
instructions at www.thermoscientific.com/pcrwebtools to
determine the Tm values of primers and optimal annealing
temperature. As a basic rule, for primers > 20 nt, anneal for
5 seconds at a Tm +3 °C of the lower Tm primer. For primers
≤ 20 nt, use annealing temperature equal to the Tm of the
lower Tm primer. In some cases, it may be helpful to use a
temperature gradient to find the optimal annealing
temperature for each template-primer pair combination. The
annealing gradient should extend up to the extension
temperature (two-step PCR). Two-step cycling without an
annealing step is recommended for high-Tm primer pairs
(Tm at least 69−72 °C).
6.3 Extension
The extension is performed at 72 °C. The recommended
extension time is 20 seconds for amplicons ≤1 kb, and 20
s/kb for amplicons >1 kb.
7. Control reactions
7.1. Direct PCR control reaction using the control
primer mix
We recommend performing direct PCR control reactions
with both direct and dilution protocols using the control
primers supplied with this kit. As a template, use the
same plant material as in the actual experiment. If the
PCR using control primer mix is not working, the plant
sample may not be suitable for direct PCR. Control
primers are supplied as a mix of primers in H
2
O that
amplify a 297 bp fragment of a highly conserved region of
chloroplast DNA. The control primer mix has been
validated with a large number of species (refer to the list
of tested plants at www.thermoscientific.com/directpcr).
Each primer concentration is 25 µM.
Primer #1 (20-mer)
5’- AGTTCGAGCCTGATTATCCC -3’
Melting point: 62.4 °C
Primer #2 (20-mer)
5’- GCATGCCGCCAGCGTTCATC -3’
Melting point: 75.5 °C
Table 3. Pipetting instructions for control reactions.
2
Buffer
10 µL 1X
Polymerase
0.4 µL
(see Section 4)
Direct protocol:
Dilution protocol
diameter leaf
sample/small
sample of seed
0.5 µL
Table 4. Cycling instructions for control reactions.
Cycle step Temp. Time Cycles
denaturation
98 °C 5 min 1
Annealing
Extension
62 °C
72 °C
5 s
20 s
40
Final Extension
4 °C
hold
1
Figure 1. Amplification of the 297 bp control DNA
fragment directly from Capsicum, maize, Arabidopsis and
tomato leaves. A 0.50 mm puncher was used to cut a
sample disc from the plant leaves. The samples were
placed directly into 20 µL PCR reactions. After PCR,
5 µL of loading buffer was added to the reaction and
15 µL was used for gel electrophoresis. + denotes the
control reaction with purified plant DNA and – is the
no-template control.
7.2. Positive control reaction with purified DNA
When optimizing the reactions, it is recommended to
perform a positive control with purified DNA to ensure
that the PCR conditions are optimal. If the positive control
with purified DNA fails, the PCR conditions should be
optimized before continuing further (see Section 8).
7.3. Negative control
It is recommended to add a no-template control to all
direct PCR assays. To monitor the efficiency of cleaning
the puncher, the cleaned sampling tool can be dipped
into the negative control sample. A second negative
control performed without dipping the puncher is
recommended to control other sources of contamination.
References
1. Demesure B. et al. (1995) Molecular Ecology 4:
129–131.
Technical support
EMEA: ts.molbio.eu@thermofisher.com
North America, Latin America & APAC: ts.molbio@thermofisher.com
NOTICE TO PURCHASER
• The purchase price of this product includes a limited, nontransferable
license under U.S. and foreign patents owned by BIO-RAD
Laboratories, Inc., to use this product. No other license under these
patents is conveyed expressly or by implication to the purchaser by the
purchase of this product.
• This product is sold under license from Affibody AB, Sweden.
8. Troubleshooting
No product at all or low yield
General
If the positive control with
purified DNA using your own
primers is not working:
• Optimize annealing
temperature.
• Make sure the cycling
protocol was performed as
recommended.
• Check primer design.
Direct protocol
If the positive control with
purified DNA and the Direct
PCR control reaction with
control primers are working, but
the actual samples yield no
product:
• Increase the PCR reaction
volume to 50 µL or use a
smaller sample (e.g. 0.35
mm punch).
• Increase the number of
cycles.
• Use dilution protocol for
amplification of large or
difficult samples and long
DNA fragments.
Dilution protocol
If the positive control with
purified DNA and the Direct
PCR control reaction with
control primers are working,
but the actual samples yield no
product:
• Dilute the supernatant
(from the sample treated
with Dilution Buffer) 1:10
and/or 1:100 with H
2
O/TE
buffer, and use 0.5 µL as a
template in PCR.
• Try both crushing and not
crushing the sample.
• Incubate the sample in
Dilution Buffer for 3 min
at room temperature and
use 0.5 µL as a template
(1:1 and 1:10 dilution) in a
20 µL reaction.
• Use smaller sample size or
increase the volume of
Dilution Buffer.
High molecular weight smears
Make sure the extension
time used was not too long
(>20 s/kb).
• Reduce the total number of
cycles.
• Increase annealing
temperature or perform a
temperature gradient PCR.
Decrease primer
concentration.
• Make sure the cutting tools
were properly cleaned with
2% sodium hypochlorite.
Include a negative control.
Low molecular weight discrete bands
Increase the annealing
temperature or perform a
temperature gradient PCR.
• Shorten extension time.
• Decrease primer
concentration.
Reduce the total number of
cycles.
• Design new primers.
• Make sure the cutting tools
were properly cleaned with
2% sodium hypochlorite.
Include a negative control.
PRODUCT USE LIMITATION
This product is developed, designed and sold exclusively for research
purposes and in vitro use only. The product was not tested for use in
diagnostics or for drug development, nor is it suitable for administration to
humans or animals. Please refer to www.thermoscientific.com/onebio for
Material Safety Data Sheet of the product.
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