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  7. Thermo Scientific™ Phire Tissue Direct PCR Master Mix

Thermo Scientific™ Phire Tissue Direct PCR Master Mix

Catalog No. F170S
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F170S 250 Reactions
F170L 1250 Reactions
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Catalog No. F170S Supplier Thermo Scientific™ Supplier No. F170S
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Description

Thermo Scientific Phire Tissue Direct PCR Master Mix has been developed for amplification of DNA directly from a wide variety of tissues obtained from mice, human, fish, birds and insects.

Thermo Scientific Phire Tissue Direct PCR Master Mix has been developed for amplification of DNA directly from a wide variety of tissues obtained from mice, humans, fish, birds, and insects. The Master Mix containing Phire Hot Start II DNA Polymerase is specially formulated to perform PCR in the presence of different animal tissue–derived inhibitors such as collagen, melanin and eumelanin (hair, skin), and myoglobin (muscle). The kit also includes DNARelease Additive, which can be used to improve the release of DNA from difficult tissues.

Features

  • Direct PCR reduces time-consuming and expensive DNA purification steps
  • Available Dilution & Storage protocol allows multiple PCR reactions from a single sample
  • Specially engineered Phire DNA polymerase for extremely short PCR protocol times
  • Master mix format with premixed gel loading dye minimizes cross-contamination, reduces sample handling, and allows direct loading on gel

Applications

  • Genotyping
  • Transgene detection
  • Knockout analysis
  • Sequencing

Notes

  • The optimal annealing temperature for Phire DNA Polymerases may differ significantly from that of Taq-based polymerases.

Specifications

Concentration 2X
Content And Storage • 2X Phire Tissue Direct PCR Master Mix, 2 x 1.25 mL
• Dilution Buffer, 5 mL
• DNARelease Additive, 3 x 100 μL
• 25 μM Control Primer Mix, 40 μL
• Nuclease-free water, 2 x 1.25 mL
• 50 μg O'GeneRuler Express DNA Ladder

Store at -20°C.
GC-Rich PCR Performance Medium
PCR Method Direct PCR
Polymerase Phire Hot Start II DNA Polymerase
Reaction Speed Fast
Includes Label or Dye Yes
Product Type Direct PCR Master Mix
Quantity 250 reactions
Shipping Condition Wet or Dry Ice
For Use With (Application) Genotyping, Hot-start PCR
Hot Start Built-In Hot Start
No. of Reactions 250 Reactions
Overhang Blunt
Reaction Format SuperMix or Master Mix
Sample Type Tissue
Starting Material DNA
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Frequently Asked Questions (FAQs)

What is your recommendation to take a sample from plant or animal tissue for Direct PCR?

In order to obtain best results from Direct PCR, it is important to use a very small amount of starting materials. The recommended size is 0.3-0.5 mm in diameter. We recommend using Tissue Puncher 0.3 mm (Cat. No. F-200S), which is a convenient tool for sampling and is suitable for both plant and animal tissues.

Why are Control primers provided in the Direct PCR Kits?

It is very important to include a control reaction in the Direct PCR to ensure that the PCR conditions are optimal for the specific tissue or plant material used. Control primers are universal primers that amplify a small fragment from a conserved genome region. If the reaction with control primers fails, the sample material may not be suitable for Direct PCR, or PCR conditions need optimization.
How are samples, stored at different conditions, compatible with the Phire Tissue Direct PCR Master Mix?

With the Phire Tissue Direct PCR Master Mix you can amplify DNA from differently stored tissues (fresh, frozen, FFPE) from human and a variety of animal species.
Can I use plant or tissue sample which is not on the list of the Direct PCR Master Mix and Kit tested samples?

You can test any plant or tissue sample. However, it is recommended to first optimize the reaction. When working with new sample materials, start with the Dilution & Storage protocol as it allows several PCR reactions to be performed from the same sample during optimization. It is recommended to perform a positive control with purified sample DNA to ensure that the PCR conditions are working. If the positive control with purified DNA fails, the PCR conditions should be optimized before continuing further.

Can you amplify the same sample materials with the Direct PCR Master Mixes as with Direct PCR Kits?

Yes, the Direct PCR Master Mixes are upgrades of the Direct PCR kits and thus are suitable for the same sample materials.
Do the Direct PCR Master Mixes and Kits come with tissue punchers?

No, tissue punchers are not provided. You can purchase 0.3 mm Tissue Punchers (Cat. No. F-200S), or cut the sample with a scalpel. Some scientists use 100 µL pipette tips to take a sample.
Do the Direct PCR Master Mixes work in Dilution & Storage protocol?

Yes, similar to the Direct PCR Kits, Direct PCR Master Mixes work in both Direct and Dilution & Storage protocols. Direct PCR from blood is performed with Direct protocol only.
Which FFPE sample types are suitable for Direct PCR from FFPE tissues?

FFPE tissue, FFPE tissue sections, tissue blocks and microscope slides can all be used in sample preparation.
What if I want to clone or sequence the PCR product from a Direct PCR kit? Can I use the kit with Phusion instead of Phire?

Direct PCR Kits and Master Mixes employ Phire Hot Start II DNA Polymerase or Phusion Hot Start II Polymerase (depending on the kit), that both possess the following activities: 5'-3' DNA polymerase activity and a weak 3'-5' exonuclease activity. When cloning fragments amplified with these DNA Polymerases, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product. However, before adding the overhangs, it is very important to remove all the DNA polymerase by purifying the PCR product carefully. Any remaining Phusion or Phire DNA Polymerases will degrade the A overhangs, creating blunt ends again. All Direct PCR kits are compatible with sequencing techniques. The green dye present in the Direct Master Mix kits does not interfere with the sequencing reactions.
I am testing your Direct PCR protocol using mouse tissue as sample material. I cannot see a PCR product in the agarose gel, but there is a big block of DNA stuck in the wells. What can I do?

When amplifying DNA directly from mouse tissue, it is important to add DNARelease Additive before gel electrophoresis as otherwise cell debris present in the PCR products can cause DNA fragments to get trapped in the agarose gel wells. Add 1.5 µL of DNARelease Additive into a 50 µL PCR reaction. If you are using Phire Animal Tissue Direct PCR Kit add the DNARelease Additive to the loading dye for gel electrophoresis. Mix 100 µL of DNARelease Additive with 1 mL of gel loading dye. The dilution is stable for 2 weeks at +4 degrees C. For long term use, store the dilution at -20 degrees C. Before agarose gel electrophoresis, add 15 µL of the pre-mixed gel loading dye into a 50 µL PCR reaction.
After performing Direct PCR, how should I store the PCR products amplified directly from the sample?

After PCR, it is recommended to centrifuge the PCR reactions at 1000 x g for 1-3 minutes to collect the supernatant for subsequent analysis. We recommend storing the supernatant at -20 degrees C
Are there any restrictions to the size of the amplicon in Direct PCR?

The optimal length for Direct PCR from liquid samples is up to 5 kb and from solid samples up to 1 kb. However, the maximum length depends on the sample type, sample size and the reaction volume. For example we have amplified a 7.5 kb fragment directly from human blood and tissue samples, and a 3 kb fragment from mouse ear and 5 kb fragment from plant leaves.
When performing Direct PCR, when do I need to use 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 and should be added directly into the PCR reaction. Please reduce the amount of water used or simply add the DNARelease to the PCR reaction setup. DNARelease is also used in the Dilution & Storage protocol to improve the release of DNA from the tissue sample.
Do you offer an alternative to the discontinued VersaTaq Direct PCR Polymerase (Cat. No. 71002)?

Yes, we recommend using Phire Tissue Direct PCR Master Mix (Cat. No. F170S, F170L).

For Research Use Only. Not for use in diagnostic procedures.