Taq dna polymerase is a thermostable enzyme that synthesizes dna from singlestranded templates in the presence of dntps and a primer. Product information polymer taq dna polymerase of reactions plus one extra. L reaction and can be used as a starting point for reaction optimization. Gotaq flexi dna polymerase is a proprietary formulation of taq dna polymerase that gives robust amplification equal to and in some cases superior to that of standard taq. Using the modified cycling protocol described here, herculase ii fusion dna polymerase also provides. Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification. The polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Aug 20, 2016 the taq dna polymerase gene is subcloned from pttq18 vector into pet28b vector and the data shows a great increase of the level of the protein expression. The supplied 5x green and colorless flexi reaction buffers do not contain magnesium. Find additional protocols for other polymerases or advanced pcr techniques in the protocols section of our pcr technologies guide. This is a basic pcr protocol using taq dna polymerase. The polymerase chain reaction pcr was developed by chemist kary mullis in the 1980s, as a means to make many copies of dna fragments.
Taq polymerase, being thermostable, proved ideal for pcr. Mytaq dna polymerase is a high performance polymerase that exhibits more robust amplification than other commonly used polymerases, delivering very high yield over a wide range of pcr templates and making it the ideal choice for most pcr assays. Pcr protocol for onetaq dna polymerase m0480 protocols. Onehalf of the total reaction volume is usually sufficient. Herculase ii fusion dna polymerase technical services us and. Pcr products generated with platinum taq dna polymerase may be used in the same downstream applications without protocol modifications. These are available online in convenient and compact pdf. Hot start pcr protocol taq dna polymerase is withheld until the reaction temperature is at 80c, to ensure high specificity.
Pcr protocol for taq dna polymerase with standard taq buffer m0273 overview. The onetaq reaction buffers and high gc enhancer have been formulated for robust yields with minimal optimization, regardless of a templates gc content. The final mgcl2 concentration may need to be optimized to account for differences in buffer formulation. Learn more about standard pcr, including what it is, on our pcr basics page. The following guidelines are provided to ensure successful pcr using nebs taq dna polymerase. Platinum taq dna polymerase high fidelity protocol 20142platinum taq dna polymerase high fidelity protocol the example pcr procedure below shows appropriate volumes for a single 50l reaction. Pcr master mix certificate of analysis 9pim750 promega. Scientists realized that thermostable heatstable dna polymerases would be needed for pcr to work efficiently. May 22, 2012 for example, taq dna polymerase has an optimal elongation temperature of 70 c to 80 c and requires 1 minute to elongate the first 2 kb, then requires an extra minute for each additional 1 kb amplified. Pfu dna polymerase is another thermostable enzyme that has an optimal elongation temperature of 75 c. The enzyme is a taq dna polymerase with buffers designed for enhanced amplification. Product information polymer taq dna polymerase of reactions.
This abbreviated protocol is provided for your convenience, but is not intended for firsttime users. Therefore, during the extension step, allow approximately 2 minutes for every 1kb to be amplified minimum extension time of 1 minute. A protocol for gotaq flexi dna polymerase which provides robust amplification equal to and in some cases superior to conventional taq dna polymerase. Common routes for contamination include using the same pipette to set up a reaction and load. Taq dna polymerase is a thermostable enzyme derived from. Pcr protocol for taq dna polymerase with standard taq buffer. Use platinum taq dna polymerase for the amplification of dna from complex genomic, viral, and plasmid templates, as well as in rtpcr. The taq dna polymerase is the most successful application of a product derived from an extremophile, with annual sales around half a billion dollars podar and reysenbach, 2006. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. Platinum taq dna polymerase is a recombinant taq polymerase complexed with a proprietary antibody that blocks the polymerase activity at ambient temperatures. A protocol for the use of gotaq dna polymerase with 5x green gotaq reaction buffer and 5x colorless gotaq reaction buffer. Taq dna polymerase is an enzyme widely used in pcr 2.
Taq polymerase is a thermostable dna polymerase i named after the thermophilic eubacterial microorganism thermus aquaticus, from which it was originally isolated by chien et al. If necessary, the reaction mixture can be overlaid with mineral oil or paraffin melting temperature 5060c of special pcr grade. Taq dna polymerase is a highly thermostable dna polymerase of the thermophilic bacterium thermus aquaticus. The 5x gotaq green and colorless reaction buffers supplied with gotaq dna polymerase contain mgcl 2 at a concentration of 7. Pluthero 1993 rapid purification of highactivity taq dna polymerase. This product is supplied with 10x reaction buffer containing 15 mm magnesium chloride.
For multiple reactions, prepare a master mix of components common to all. The extension rate of pfudna polymerase is lower than that of taq dna polymerase. The data shows that the purified protein product contains very few heteroproteins, indicating that its purity andor activity level would be high and in favor of the pcr reaction. Purification of taq dna polymerase for 1 liter culture modified from the protocol presented in f. The key to the polymerase chain reaction was first discovered in1976. Activity is restored after the initial denaturation step in pcr cycling at 94c, providing an automatic hot start and offering increased sensitivity, specificity, and yield. Day 1 start 3ml overnight culture of taq from glycerol stock in lbamp 75ngul day 2 add 1ml of overnight culture to 1l of lbamp. Contamination can be a major problem, unless you are careful. Arguably one of the most powerful laboratory techniques ever discovered, pcr combines the unique attributes of being very sensitive and specific with a great degree of flexibility.
The polymerase chain reaction pcr is the cardinal laboratory technology of molecular biology. It has a 53 dna polymerase activity and a 53 exonuclease activity. Gotaq dna polymerase is a proprietary formulation of taq polymerase that gives robust amplification equal to and, in some cases, superior to that of standard taq. Taq dna polymerase is a thermostable dna polymerase isolated from an e. Home protocols pcr protocol for taq dna polymerase with standard taq buffer m0273 pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. Protocol to prepare several parallel reactions and to minimize the. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols.
Therefore, taq dna polymerase can efficiently synthesize dna under the heatintensive conditions of the pcr reaction. Taq dna polymerase, from thermus aquaticus recombinant. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. Titanium taq can be used in a wide variety of pcr applications, and the constraints on. Titanium taq pcr kit protocolataglance pt33042 please read the user manual before using this protocolataglance. Datasheet for taq dna polymerase with standard taq buffer neb. Data of expression and purification of recombinant taq dna. The following protocol is a general guideline and starting point for pcr amplification, however, as with all polymerases, optimal reaction conditions for incubation.
Themagnesium concentrationdoesnot need to be adjusted. Taq dna polymerase is the most common polymerase used for pcr. Taq dna polymerase pcr buffer this product is compatible for use in the polymerase chain reaction pcr process claimed in patents owned by roche molecular systems, inc. Taq buffer with kcl and taq buffer with nh 4 2 so 4. The key is the taq polymerase that was purified from the thermophile thermus aquaticus. Taq dna polymerase this dna polymerase is isolated from the bacterium thermus aquaticus, which lives in hot environments and requires biomolecules that are heat stable. Taq dna polymerase is an enzyme widely used in pcr. Allows optimization of mgcl 2 concentration and facilitates directtogel analysis of pcr products. Herculase ii fusion dna polymerase provides accuracy comparable to pfu dna polymerase. Pcr protocol for taq dna polymerase with standard taq buffer m0273. Always check your primer design before constructing or ordering primers. Moreover, the special enzyme formulation and optimized buffer system ensure robust performance when amplifying difficult and gcrich targets. Cycling conditions amplification parameters depend greatly on the template, primers and amplification apparatus.
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