Thermo Scientific™ Phusion Blood Direct PCR Kit

Catalog No.	No. of Reactions
F547L	500 Reactions
F547S	100 Reactions

Catalog No. F547L Supplier Thermo Scientific™ Supplier No. F547L

Description

Thermo Scientific Phusion Blood Direct PCR Kit is developed for amplification of DNA from whole blood. It eliminates the need for a separate DNA purification step prior to PCR.

Thermo Scientific Phusion Blood Direct PCR Kit is for use in the amplification of DNA from whole blood. It eliminates the need for a separate DNA purification step prior to PCR. The modified Phusion Hot Start II High-Fidelity DNA Polymerase exhibits high resistance to PCR inhibitors found in blood and retains polymerase activity even with samples containing 40% whole blood.

This DNA polymerase exhibits exceptional accuracy in proofreading and makes the Phusion Blood Direct PCR Kit one of the best choices for demanding applications, such as cloning. The Phusion Blood Direct PCR Kit is versatile and permits analysis of blood samples from various species and with different anticoagulants or storage conditions.

Features

• No need for time-consuming and expensive DNA purification steps
• Simple protocol requires minimal hands-on time
• Extremely short PCR protocol times
• Modified Phusion Hot Start II High-Fidelity DNA Polymerase delivers abundant yields of specific product

Notes

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

Specifications

• Concentration: 2 U/μL
• Content And Storage: • Phusion Blood II DNA Polymerase, 200 μL
  • 2X Phusion Blood PCR Buffer with dNTPs and MgCl₂, 5 x 1.25 mL
  • Universal Control Primer Mix, 40 μL
  • 50 mM EDTA, 0.5 mL
  • 50 mM MgCl₂, 1.5 mL
  • DMSO, 0.5 mL
  
  Store at -20°C.
• GC-Rich PCR Performance: High
• PCR Method: Direct PCR
• Polymerase: Phusion Blood II DNA Polymerase
• Reaction Speed: Fast
• Product Type: Direct PCR Kit
• Quantity: 500 reactions
• Shipping Condition: Wet or Dry Ice
• Hot Start: Built-In Hot Start
• No. of Reactions: 500 Reactions
• Overhang: Blunt
• Reaction Format: Separate Components
• Sample Type: Blood
• Starting Material: DNA

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.

Can additional markers be added to Thermo Scientific Animal Genotyping kits?

No, extra markers cannot be added to any of the Animal Genotyping kits. All primer mixes are optimized to work in a multiplex PCR. Addition of one or more primers would require optimization of the primer concentrations and the PCR protocol.

Are primers from Thermo Scientific Animal Genotyping kits available separately for specific loci?

We do not provide primers for specific locii separately. They come only in a multiplex format. If one or several loci did not amplify from the sample, faint signals can be a result of low DNA amounts or impurities. Some loci may be more sensitive towards purity of DNA used and may vary from sample to sample. On rare occasions, an animal can have a deletion in the marker region.

Do Thermo Scientific Animal Genotyping kits include an allelic ladder?

No, none of the genotyping kits contain an allelic ladder. Animal STR loci are highly polymorphic. Each allele has too many variants to make a universal allelic ladder.

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.

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.

What is the highest blood concentration possible in the reaction when using Phusion Blood Direct PCR Master Mix and Kit?

We recommend using blood concentration up to 20%; however, we had successful amplifications with up to 40% of blood in the reaction mix. In some cases optimization may be required to achieve this.

Why is EDTA included in the Phusion Blood Direct PCR Master Mix and Kits?

Extensive testing has shown 3.0 mM MgCl2 to be optimal concentration for most PCR reactions using whole blood. There is a separate tube of 50 mM MgCl2 (F-510MG) included in the kit for adjusting the MgCl2 concentration (up to 4.5 mM if necessary) for reactions containing very high amounts of blood. It should be noted though that excess MgCl2 may result in spurious PCR products. If unspecific products are created with the default reaction buffer (providing 3.0 mM MgCl2), the effective MgCl2 concentration can be decreased by adding the chelating agent EDTA, which is included in the kit. Typically, adding 0.5 to 1.0 µL of 50 mM EDTA to a 20 µL reaction is sufficient to eliminate non-specific products. Note that the optimal conditions will depend on the primers, the percentage of blood in the reaction and/or the type of card used, since anticoagulants and other chemicals impregnating the cards can alter the available Mg2+ concentration.

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.

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.

Does Phusion Blood Direct PCR Kit and Master Mix have an IVD-CE mark?

No.

Can I use Phusion Blood Direct PCR Kit/Master Mix for allele-specific PCR directly from blood (e.g. HLA-typing)?

Phusion Blood DNA Polymerase has a strong 3'- 5' exonuclease activity which is able to cleave the mismatched base from the 3' end and continue amplification. If the mismatch is located at the 3' end and is not protected from cleavage by any modification, Phusion polymerase will cut the mismatched base.

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.

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