New England Biolabs, M0500S, Q5® Blood Direct 2X Master Mix

Related Categories Master Mixes,, PCR, qPCR & Amplification Technologies Applications Extraction-Free PCR,, qPCR & RT-qPCR,, Two-Step RT-qPCR, Specification Heat Inactivation No FAQ Q: What is the difference between Q5® Blood Direct 2X Master Mix and Hemo KlenTaq (NEB #M0332)? A: 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. Q: What types of samples/materials are compatible with the Q5® Blood Direct 2X Master Mix? A: 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. Q: What concentration of whole blood is recommended for use with Q5® Blood Direct 2X Master Mix? A: 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 Q: What blood anticoagulants can be used with Q5® Blood Direct 2X Master Mix? A: K2 EDTA, Na EDTA, Na citrate and Na heparin anticoagulants are all compatible with Q5 Blood Direct 2X Master Mix. Q: How do I pre-treat Dried Blood Spot Punches? A: Pre-treatment is generally not necessary for a single 0.5 mm punch in a 20 µL reaction or a single 1 mm punch in a 50 µL reaction. However, when using more or larger punches, they can be pre-treated to avoid possible inhibition of PCR. To pre-treat, place a single punch in a PCR tube. Add 50 µL of nuclease-free water and heat to 50°C for 5 minutes. Remove the supernatant with a pipette tip and use the washed punch in the PCR reaction. Q: What length of product can be made by Q5® Blood Direct 2X Master Mix? A: From human blood cells, we have amplified products up to 7.5 kb. Q: What ends will my PCR products have? A: APPLICATION POLYMERASE PRODUCTS PCR PRODUCT ENDS High fidelity PCR Q5® polymerases Blunt Phusion® polymerases Blunt Routine & Specialty PCR OneTaq® polymerases 3'A/blunt Taq polymerases 3'A LongAmp® polymerases 3'A/blunt Hemo KlenTaq Polymerase 3'A Isothermal amplification Bst polymerases 3'A Bsu Polymerase 3'A phi29 Polymerase Blunt DNA manipulation T7 DNA Polymerase Blunt E. coli DNA Polymerase I Blunt DNA Polymerase I, Large (Klenow) Fragment Blunt Klenow Fragment (3′-5′ exo-) 3'A T4 DNA Polymerase Blunt Vent® Polymerase Blunt Vent® (exo-) Polymerase 3'A Deep Vent® Polymerase Blunt Deep Vent® (exo-) Polymerase 3'A For more details about our polymerases, including exonuclease activities and applications, please visit our DNA Polymerase Selection Chart. Learn More For more information about exonuclease activity, check out this FAQ. Why do some polymerases blunt and others add a nucleotide? Polymerases that possess proofreading (3´-5´ exonuclease) activity, such as Q5, Phusion, and Deep Vent, will add an untemplated nucleotide to the 3' ends of extended DNA fragments, but the exonuclease activity subsequently removes it. Other polymerases that lack 3´-5´ exonuclease activity (such as Taq and Taq-based polymerases) will add an extra nucleotide to 3´ ends (predominantly, but not exclusively, dA) and leave the untemplated overhang intact. This is why it is important to know which polymerase to use when performing blunt-end or T/A cloning. OneTaq and LongAmp Taq DNA polymerases are optimized blends of Taq (a Family A polymerase) and Deep Vent (a Family B polymerase) DNA Polymerases. The intrinsic polymerase activity of Taq adds a non-templated 3´A, while the 3´–5´ exonuclease activity of Deep Vent increases the fidelity and robustness of Taq, but also blunts PCR products. This is why these products produce a mixture of DNA ends. However, the majority of ends will have a 3'A overhang. Q: What are Hot Start and WarmStart® polymerases and when would I use them? A: When setting up room temperature reactions off ice When non-specific amplification is observed What does Hot Start/ Warm Start mean? Our Hot Start and WarmStart polymerases utilize aptamers that inhibit enzyme activity at room temperature, which discourages the formation of nonspecific products. The presence of these aptamers does not alter core enzyme function. The distinction between Hot Start and WarmStart is that Hot Start enzymes are thermophilic while WarmStart® enzymes are mesophilic; the thermodynamic range of the aptamers for Hot Start and WarmStart enzymes are largely similar. Aptamers are engineered oligonucleotides that bind to a specific target molecule through non-covalent interactions and include specific nucleobase modifications that can improve inhibition profiles and/or reduce unintended side effects. The benefit of aptamer-based inhibition over other Hot Start technologies (like antibodies) is that they do not require an activation step and bind reversibly in a temperature-dependent manner. NEB offers polymerases with aptamers for routine PCR, high-fidelity PCR, isothermal amplification, and reverse transcription. These aptamers can target different enzymatic functions for different polymerases. Learn More Why is it called "Hot Start?" Like many non-proofreading, Family A DNA polymerases, Taq Polymerase possesses the ability to add bases onto the end of ssDNA in a template-independent manner even at room temperature, and can result in the addition of non-specific bases onto the ends of DNA primers in the reaction, enabling off-target hybridization and reduced overall reaction specificity. In contrast, at higher temperatures, nonspecific binding is reduced as annealing becomes more stringent. Early methods to mitigate undesired activity at low temperature focused on the exclusion of key reaction components until the reaction temperature was increased, which could then be spiked into the mixture, triggering the reaction under a more restrictive, high temperature condition. Read our feature article, Using aptamers to control enzyme activities Hot Start Taq and beyond, to see data comparing product formation for enzymes with and without aptamer-based inhibition of activity. Q: What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)? A: POLYMERASE PRODUCTS FIDELITY* ERROR RATE PRODUCT ENDS MAX PRODUCT LENGTH** EXTENSION TEMPERATURE MODIFIED NUCLEOTIDE INCORPORATION*** URACIL INCORPORATION 5´-3´ EXONUCLEASE 3´-5´ (PROOFREADING) EXONUCLEASE Q5 Polymerases 280X <0.44 x 10-6 Blunt 20kb simple, 10kb complex 72°C 5mC, 5hmC, 6mA No (except Q5U) - ++++ Phusion Polymerases 39-50X 0.44 x 10-6 Blunt 20kb simple, 10kb complex 72°C 5mC, 5hmC No - ++++ OneTaq Polymerases 2X <140 x10-6 3´A/blunt 6kb 68°C 5mC, 5hmC, biotin, DIG Yes + ++ Taq Polymerases 1X 2.85 x 10^-4 3´A 5kb 68°C 5mC, 5hmC, biotin, DIG Yes + - Hemo KlenTaq nt nt 3´A 2kb 68°C Yes No - LongAmp® Polymerases 2X 3´A/blunt 30kb 65°C No Yes ++ *Fidelity relative to Taq DNA polymerase. We continue to investigate assays to characterize Q5's very low error rate to ensure that we present the most accurate fidelity data possible (Potapov, V, and Ong, J.L. (2017) PloS ONE, 12(1): e0169774). **Simple templates include plasmid, viral and E. coli genomic DNA. Complex templates include plant, human and other mammalian genomic DNA and cDNA. *** For more information, contact Technical Support at info@neb.com For more information on properties to help you select a polymerase for your application, please see our DNA Polymerase Selection Chart. Learn More Fidelity and error rate The fidelity of a DNA polymerase is defined by its ability to accurately replicate a template, while error rate is the rate of misincorporation of an incorrectly matched nucleotide. Fidelity is important for applications in which the DNA sequence must be correct after amplification. To learn more about how fidelity is measured, click here. Product ends and exonuclease activity Check out the "Learn More" section on our PCR Product Ends FAQ and our Exonuclease Activity for DNA Polymerases FAQ. Q: My results are not as expected. Where can I find troubleshooting help? A: Nonspecific amplification, no amplification, wrong product size Curious result? Consult our PCR Troubleshooting Guide after your reaction to identify potential causes of unexpected results and solutions. More details on reaction conditions and setup optimization can be found in our Guidelines for PCR Optimization with Thermophilic DNA Polymerases and this blog post. Technical Support is always happy to work with you to troubleshoot your PCR. If you would like assistance, you can: Email us at info@neb.com Call Technical Support at (800)-0632-7799, available Monday through Friday, 9:00AM - 6:00PM EST Fill out this webform Failure to amplify a target greater than 5 kb If you are struggling to amplify a target that is greater than 5 kb, try some of these tips: We recommend using Q5®, Phusion®, or LongAmp® polymerases If using Q5, try decreasing the final primer concentration to 150-300nM Stand-alone enzyme + buffer formulations allow more flexibility in reaction optimization than master mixes Use more template Treat the purified template gently as not to shear it Optimize enzyme concentration by testing a titration of enzyme in the reaction (0.25-2 units/50μl reactions) Increase the number of cycles Lengthen extension time to 40s/kb Smearing on an agarose gel When PCR conditions are not optimal, a smear or high level of background is often observed. Try one or more of the following suggestions: Use less enzyme Decrease the extension temperature to 3°C below the extension temperature recommended by the specific product protocol For example, the OneTaq® protocol recommends a 68°C extension temperature; try 65°C. Raise the annealing temperature Try 2-step cycling protocols If there is an illuminated halo around the well in addition to smearing from the well, use less template. Q: When and how should I use the Q5® High GC Enhancer? A: The Q5 High GC Enhancer is an additive that should be used when dealing with particularly difficult or high GC templates, but can be inhibitory when using high AT content templates. The stand-alone Q5 enzyme can cover a wider range of GC content (up to 80%) with the addition of the GC enhancer. Q5 High GC Enhancer is a supplemental reagent to Q5 enzyme and buffer formulations (M0491 and M0493) and should not be used alone. *This Enhancer is not a stand-alone buffer and should not be used on its own. Additionally, it should not be added to any Q5 Master Mixes (M0492, M0494, M0500, E0555). Q5U does not benefit from the Q5 High GC Enhancer and we do not recommend using it. The addition of common PCR additives, such as up to 2% DMSO, may improve amplification of certain difficult or long targets. It is often not necessary to alter the annealing temperature of your reaction after adding Q5 High GC Enhancer. We recommend using our Tm calculator to determine the annealing temperature of your PCR. Learn More Use of the Q5 High GC Enhancer often lowers the effective range of temperatures at which specific amplification can be observed by reducing complex template secondary structures, which can increase the amplification of your target DNA and improve your yield of hard to amplify products, such as GC rich templates. Generally, PCR additives usually work one of two ways: By reducing secondary DNA structures, thus increasing the amplification of your target DNA Secondary DNA structures can be destabilized by additives that bind to the minor and major grooves of DNA and affecting hydrogen bonding of the duplex. Secondary structures include the double helix (increased hydrogen bonding due to increased GC content) and stem-loop structures (hairpins or bulging nucleotides that reduce hybridization) By reducing non-specific priming and thus reducing the amplification of off-target DNA.