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PRODUCT INFORMATION

Thermo Scientific

Phire Tissue Direct

PCR Master Mix

#F-170S 100 rxns

Lot 00000000 Expiry Date ____

Danger.

Hazard statements:

May cause allergy or asthma symptoms or breathing

difficulties if inhaled.

Precautionary statements:

In case of inadequate ventilation wear respiratory

protection. Avoid breathing dust/fume/gas/mist/vapours/spray. If

experiencing respiratory symptoms: Call a POISON CENTER or

doctor/physician. IF INHALED: If breathing is difficult, remove victim to

fresh air and keep at rest in a position comfortable for breathing. Dispose

of contents/container in accordance with

local/regional/national/international regulations.

Thermo Fisher Scientific Baltics UAB, V.A. Graiciuno 8, LT-02241 Vilnius,

Lithuania, tel. +370 700 55131.

Store at -20°C

Extended version of product information is available online

www.thermoscientific.com/directpcr

www.thermoscientific.com/onebio

COMPONENTS OF THE PRODUCT

Component

#F-170S

50 rxns x 20 µL

100 rxns x 50

µL

#F-170L

1250 rxns x 20

µL

500 rxns x 50

µL

2X Phire Tissue Direct

PCR Master Mix

2 × 1.25 mL 10 × 1.25 mL

Dilution Buffer 5 mL 2 × 12.5 mL

DNARelease Additive 3 × 100 µL 1.3 mL

Universal Control

Primer Mix

(25 µM each)

40 µL 40 µL

Water, nuclease- free 2 × 1.25 mL 10 × 1.25 mL

O’GeneRuler Express

DNA Ladder

100 applications

(50 µg)

Shipping and storage

Upon arrival, store the components at −20 °C. The

Dilution Buffer can also be stored at 4 °C once it is

thawed.

Rev.5

1. Introduction

Thermo Scientific™ Phire™ Tissue Direct PCR Master

Mix is designed to perform PCR directly from tissue

samples with no prior DNA purification. Tissues such as

mouse ear and tail, zebrafish fin, Drosophila, human hair

are suitable starting materials. The samples can either

be fresh or stored at −70 °C. A list of tissues tested with

this Master Mix is available at

www.thermoscientific.com/directpcr.

The Phire Tissue Direct PCR Master Mix contains

reagents for two alternative protocols: Direct and Dilution

& Storage protocols. The Master Mix is recommended for

end point PCR protocols and it contains premixed gel

loading dye which allows direct sample loading on the

gel. The loading dye in the Master Mix does not interfere

with PCR performance and is compatible with

downstream applications such as DNA sequencing,

ligation and restriction digestion.

2. Important Notes

• Detailed protocols for specific tissue samples are

available on www.thermoscientific.com/directpcr

• Primer annealing temperatures with Phire are

different from many common DNA polymerases

(such as Taq DNA polymerases). Read Section 7.3

carefully.

• Add the sample directly into a PCR reaction instead

of an empty tube.

• The Dilution & Storage protocol is recommended:

• When working with new sample materials or a

new primer pair.

• With difficult samples or long amplicons.

• When performing multiple reactions from the

same sample.

3. PCR Protocol

Before starting read all Important notes (Section 2) and

Sample handling guidelines (Section 4). The PCR setup

can be performed at room temperature. Always add the

sample last to the reaction. Read Section 4 carefully

for sampling guidelines.

Table 1. Pipetting instructions (add items in this order)

Component 20 µL rxn 50 µL rxn*

Final

conc.

O add to 20 µL add to 50 µL

2X Phire Tissue

Direct PCR Master

Mix

10 µL 25 µL 1X

Primer A X µL X µL 0.5 µM

Primer B X µL X µL 0.5 µM

Sample

Direct Protocol:

Dilution & Storage

Protocol:

–

0.5 -1 μL

Amount depends

on the sample**

1.25 - 2.5 μL

* 50 μL reaction volume is recommended for the Direct protocol.

** 0.5 mm punch or a small sample of tissue (see

www.thermoscientific.com/directpcr)

Table 2. Cycling protocol

Cycle step

2-step 3-step

Cycles

Temp. Time Temp. Time

Initial

denaturation

98 °C 5 min 98 °C 5 min 1

Denaturation

Annealing

(see 6.3)

Extension

(see 6.4)

98 °C

72 °C

5 s

20 s ≤1

20 s/kb >1

98 °C

X°C

72 °C

5 s

20 s ≤1

20 s/kb >1

Final

Extension

72 °C

+4 °C

1 min

hold

72 °C

+4 °C

1 min

hold

Gel electrophoresis

2X Phire Tissue Direct PCR Master Mix contains a

premixed gel loading dye. After PCR samples can be

directly loaded on the electrophoresis gel for analysis.

Positive control reaction with purified DNA

When optimizing the direct PCR protocol, 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 until the control PCR

gives a desired result. For troubelshooting refer to

extended product information available online.

Negative control

It is recommended to use a no-template control with all

Direct PCR assays to control contamination. To monitor

the efficiency of cleaning the sampling tool, the cleaned

tool can be dipped into the negative control sample. A

second negative control performed without dipping the

sampling tool is recommended to control for other

sources of contamination.

4. Guidelines for Sample Handling

To obtain small and uniform samples, we recommend

using 0.35 – 0.50 mm diameter puncher. If the puncher

is to be reused, it is very important to clean the cutting

edge properly to prevent cross-contamination between

samples. Use 2% NaClO solution for cleaning and cross

contamination prevention.

Other ways to take a sample is by cutting with scalpel to

obtain 0.35 – 0.50 mm sample. Scalpel must be cleaned

properly to prevent cross-contamination between

samples.

5. Choosing the Protocol

This Master Mix is optimized for various tissue samples.

Please visit www.thermoscientific.com/directpcr to

see a list of tested tissues. With a few exceptions, both

Direct and Dilution & Storage protocols are compatible

with all sample types and applications. However, when

amplifying longer fragments (e.g. > 500 bp from fish fin

tissue or > 1 kb from other tissues) the Dilution &

Storage protocol is recommended.

5.1 Direct Protocol

Direct Protocol: Take a sample of 0.5 mm in diameter

from tissue by using the puncher or use a sterile scalpel

to cut a very small piece of tissue (e.g. one Drosophila

leg). Place the sample directly into the PCR reaction

(50 μL of volume). It is recommended to place the

sample into the liquid rather than into an empty tube.

Make sure that you see the sample in the solution.

5.2 Dilution & Storage protocol

Before beginning, warm a heat block to 98 °C. Place the

tissue sample into 20 μL of Dilution Buffer. Add 0.5 μL of

DNARelease Additive. Mix by vortexing the tube briefly,

and spin down the solution. If a larger sample is used,

adjust the volume of the Dilution Buffer and DNARelease

Additive accordingly. Make sure the sample is covered

with the solution. Incubate the reaction for 2−5 minutes

at room temperature and then place the tube into the

pre-heated (98°C) block for 2 minutes. Spin down the

remaining tissue and store the supernatant at −20 °C if

not used immediately.

Usually 1 μL of supernatant is sufficient for a 20 μL PCR

reaction. In some cases the supernatant may have to be

diluted 1:10 or 1:100, or the PCR reaction performed in a

50 μL volume.

6. Notes About Reaction Components

6.1. Phire Tissue Direct PCR Master MIx

2X Phire Tissue Direct PCR Master Mix contains the

dNTPs and provides 1.5 mM MgCl

concentration in the

final reaction. The Master Mix employs Phire Hot Start II

DNA Polymerase, that possesses the following activities:

5´→3´ DNA polymerase activity and a weak 3´→5´

exonuclease activity. When cloning DNA fragments

amplified with Phire Hot Start II DNA Polymerase blunt

end cloning is recommended.

6.2. Dilution & Storage Buffer

The Dilution & Storage Buffer has been optimized to

release DNA from a wide variety of different tissues

when supplemented with DNA Release Additive (see

Section 6.3.). Samples in Dilution Buffer can be stored

for up to 4 weeks in different temperatures (-20 °C,

+4 °C and room temperature) before using in PCR. For

long term storage, it is recommended to transfer the

supernatant into a new tube and store at −20 °C.

6.3. DNARelease Additive

DNARelease Additive is required when PCR is

performed directly from certain tissue samples using the

Direct protocol. Cell debris present in these PCR

products can cause DNA fragments to get trapped in the

agarose gel wells. DNARelease Additive eliminates this

problem. DNARelease Additive is also used in the

Dilution & Storage protocol to improve the release of

DNA from the tissue sample.

Add 1.5 μL of DNARelease

Additive into a 50 μL PCR reaction.

6.4. Primers

The recommendation for the 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 on our website

www.thermoscientific.com/tmc to determine the

Tm values of primers and optimal annealing temperature.

7. Notes About Cycling Conditions

7.1. 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.

7.2. Denaturation

Keep the denaturation time as short as possible. Usually

5 seconds at 98 °C is enough for most templates. Note

that the denaturation time and temperature may vary

depending on the ramp rate and temperature control

mode of the thermal cycler.

7.3. 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 on Thermo Scientific

website 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 an annealing

temperature equal to the Tm of the lower Tm prime.

Two-step cycling without an annealing step is

recommended for high-Tm primer pairs (Tm at least

69−72 °C).

7.4. 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.

8. Control Reactions

8.1. Direct PCR control reaction using the control

primer mix

When using mammalian tissue samples (e.g. mouse,

human tissue), we recommend performing Direct PCR

control reactions with both Direct and Dilution & Storage

protocols using the control primers supplied with this

Master Mix. As a template, use the same tissue material

as in the actual experiment. The universal control primer

mix contains degenerate primers that amplify a 237 bp

fragment of mammalian genomic DNA. The amplified

region is a highly conserved non-coding region upstream

of the SOX21 gene

and the primers are designed to

amplify this region from a wide range of vertebrate

species.

Each primer concentration is 25 μM.

Primer #1 (24-mer)

5’- AGCCCTTGGGGASTTGAATTGCTG -3’

Melting point: 73.5 °C

Primer #2 (27-mer)

5’- GCACTCCAGAGGACAGCRGTGTCAATA -3’

Melting point: 72.2 °C (R=A), 75.3 °C (R=G)

Please note that these control primers are not compatible

with fish or insect samples. The recommended control

primer sequences for Drosophila and zebrafish are

available at www.thermoscientific.com/directpcr.

Table 3. Pipetting instructions for control reactions.

Component 20 µL rxn 50 µL rxn*

O add to 20 µL add to 50 µL

2X Phire Tissue Direct

PCR Master Mix

10 µL 25 µL

Universal control primer

mix

0.4 µL 1 µL

Samples

Direct Protocol:

Dilution & Storage

Protocol:

–

1 μL

Amount depends

on the sample**

2.5 μL

*50 μL volume is recommended for Direct protocol.

**0.5 mm punch or a small sample of tissue (see

www.thermoscientific.com/directpcr)

Table 4. Cycling instructions for control reactions using

primers included.

Cycle step Temp. Time Cycles

Initial denaturation 98 °C 5 min 1

Denaturation

Annealing/Extension

98 °C

72 °C

5 s

20 s

Final Extension

72 °C

4 °C

1 min

hold

CERTIFICATE OF ANALYSIS

Performance in PCR is tested by the amplification 7.5 kb

fragment from human genomic DNA.

Absorption measured at 424 nm and 614 nm.

Quality authorized by: Jurgita Zilinskiene

REFERENCES

1. Woolfe A. et al.(2005) PLoS Biology3: 116–130.

Troubleshooting

To optimize Direct PCR three key steps have to be

considered: dilution protocol, sample size and optimal

primer annealing temperature.

Troubleshooting information is available in the extended

version of the protocol. See

www.thermoscientific.com/directpcr for more details.

SAFETY INFORMATION

DNARelease Additive

Danger

Hazard statements:

H334 May cause allergy or asthma symptoms or breathing

difficulties if inhaled.

Precautionary statements:

P285 In case of inadequate ventilation wear respiratory

protection.

P261 Avoid breathing dust/fume/gas/mist/vapours/spray.

P342+P311 If experiencing respiratory symptoms: Call a

POISON CENTER or doctor/physician.

P304+P341 IF INHALED: If breathing is difficult, remove

victim to fresh air and keep at rest in a position comfortable

for breathing.

P501 Dispose of contents/container in accordance with

local/regional/national/international regulations.

NOTICE TO PURCHASER:

• Use of this product is covered by US Patent No.

6,127,155. The purchase of this product includes a

limited, non-transferable immunity from suit under the

foregoing patent claims for using only this amount of

product for the purchaser’s own internal research. No right

under any other patent claim, no right to perform any

patented method and no right to perform commercial

services of any kind, including without limitation reporting

the results of purchaser's activities for a fee or other

commercial consideration, is conveyed expressly, by

implication, or by estoppel. This product is for research

use only. Diagnostic uses under Roche patents require a

separate license from Roche. Further information on

purchasing licenses may be obtained by contacting

outlicensing@lifetech.com or Out Licensing, Life

Technologies Inc., 5791 Van Allen Way, Carlsbad,

California 92008.

• The purchase price of this product includes a limited, non-

transferable 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.

LIMITED USE LABEL LICENSE: Internal Research and

Development Use Only.

The purchase of this product conveys to the buyer the

limited, non-exclusive, non-transferable right (without the

right to resell, repackage, or further sublicense) to use this

product for internal research and development purposes.

No other license is granted to the buyer whether expressly,

by implication, by estoppel or otherwise. In particular, the

purchase of the product does not include or carry any right

or license to use, develop, or otherwise exploit this product

commercially and no rights are conveyed to the buyer to use

the product or components of the product for purposes

including but not limited to provision of services to a third

party, generation of commercial databases or clinical

diagnostics. This product is sold pursuant to authorization

from Thermo Fisher Scientific and Thermo Fisher Scientific

reserves all other rights. For information on purchasing a

license for uses other than internal research and

development purposes, please contact

outlicensing@lifetech.com or Out Licensing, Life

Technologies Inc., 5791 Van Allen Way, Carlsbad, California

92008.

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.

trademarks are the property of Thermo Fisher Scientific Inc.

and its subsidiaries.