신제품 Q5® Blood Direct 2X Master Mix는 purified DNA 대신 human whole blood (up to 30%) 또는 dried blood spots을 template로 direct PCR을 할 수 있습니다. Blood Direct 2X Master Mix에는 Q5 Hot Start High-Fidelity DNA Polymerase, dNTPs 그리고 혈액 내 inhibitors, anti-coagulants 및 여과지의 화학 물질에 저항성을 갖는 최적화된 buffer가 mix 되어 있습니다.
- Skip DNA purification and amplify direct from human whole blood cells or dried blood spots
- Utilize Q5 Hot Start High-Fidelity DNA Polymerase for accurate and efficient PCR
- Benefit from increased resistance to inhibitors and chemicals with our optimized buffer system
- Set up reactions at room temperature with aptamer-based enzyme control
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Protocol for Routine PCR Directly from Whole Blood or Dried Blood Spots (DBS) using Q5® Blood Direct 2X Master Mix (클릭하기)
| NEB # | Component Name | Amount | Concentration |
| M0500S | Q5® Blood Direct 2X Master Mix | 1 x 1 ml | 2 X |
| M0500L | Q5® Blood Direct 2X Master Mix | 5 x 1 ml | 2 X |
What concentration of whole blood is recommended for use with Q5® Blood Direct 2X Master Mix?
5-10% (v/v) whole blood is the recommended starting concentration for Q5 Blood Direct 2X Master Mix. It is possible to use up to 40% blood but cycling conditions may need to be modified. For higher blood volumes ( ≥ 20%) a 50 µL reaction volume is recommended due to difficulties in recovery of the aqueous phase from blood cell debris that remains after PCR.
What types of samples/materials are compatible with the Q5® Blood Direct 2X Master Mix?
This kit has been evaluated using up to 40% (v/v) human whole blood preserved with Na EDTA, K2 EDTA, Na citrate and Na heparin anticoagulants. However, we suggest starting with 5-10% (v/v) blood.
The Master Mix has also been tested with punches of dried human blood spotted on Whatman® 903 and FTA papers. We recommend starting with a single 0.5 mm punch in a 20 µL reaction or a single 1 mm punch in a 50 µL reaction.
What is the difference between Q5® Blood Direct 2X Master Mix and Hemo KlenTaq (NEB #M0332)?
Hemo KlenTaq is a blood direct polymerase based on a mutant Taq DNA Polymerase while Q5 Blood Direct 2X Master Mix contains Q5 Hot Start High-Fidelity DNA Polymerase.
Q5 Blood Direct 2X Master Mix comes as a mix containing buffer and dNTPs as well as enzyme. Hemo KlenTaq is a stand-alone enzyme with a separate 5X reaction buffer that does not contain dNTPs.
Q5 Blood Direct 2X Master Mix should be used in applications where a higher degree of fidelity is required, such as when the PCR products will be used in downstream applications.
Q5 Blood can amplify up to 7.5 kb fragments from human blood while Hemo KlenTaq cannot amplify fragments longer than 2 kb.
Figure 1: Q5 Blood Direct 2X Master Mix enables robust amplification direct from whole or dried blood
PCR was performed using Q5 Blood Direct 2X Master Mix under standard recommended conditions with 35 cycles of amplification. Yield and purity were quantitated by microfluidic LabChip® analysis and are indicated by dot size and color, respectively, with a large, dark green dot representing the strongest performance. A. Amplification of a variety of human genomic amplicons, 0.3 to 7.5 kb in length, from 10% EDTA-preserved human whole blood. Results are shown as both a virtual gel (top) and corresponding dot plot (bottom). Q5 Blood Direct 2X Master Mix performs well across a broad range of amplicon sizes. B. Amplification of a 604 bp human genomic amplicon from whole blood (top) or blood dried on filters (bottom). Human whole blood comprised 5-30% of the total reaction volume (50 µl) as indicated. Untreated 1 mm punches from dried blood spots were added directly to 25 µl reactions (one punch per reaction), even where pre-treatment of the punch was recommended by the manufacturer. Q5 Blood Direct 2X Master Mix shows broad tolerance to varying blood volumes, preservatives and punch types.
Figure 2: Q5 Blood Direct 2X Master Mix offers strong performance on the widest variety of targets
PCR was performed with human amplicons varying in (A) GC content (23-77%) and (B) length (2 to 7 kb), using Q5 Blood Direct 2X Master Mix and several other commercially available blood direct polymerases. 10% EDTA-preserved human whole blood was used as starting material input. 50 µl reactions were cycled according to manufacturers’ recommendations. Yield and purity were quantitated by microfluidic LabChip® analysis and are indicated by dot size and color, respectively. Q5 Blood Direct 2X Master Mix shows consistently strong performance for the broadest range of targets.
Figure 3: Consistent performance across filter types, blood preservatives and reaction volumes
PCR was performed with a 604 bp human genomic amplicon from dried blood using Q5 Blood Direct 2X Master Mix under standard recommended conditions. Whatman 903 or FTA Elute paper was spotted with blood preserved with various anticoagulants. Punches from these dried blood spots were added direct to 20, 25 or 50 µl reactions, without pre-treatment. Amplification was robust regardless of volume size, anticoagulant, or filter type.
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