Pcr products generated with platinum taq dna polymerase may be used in the same downstream applications without protocol modifications. The final mgcl2 concentration may need to be optimized to account for differences in buffer formulation. The enzyme is a taq dna polymerase with buffers designed for enhanced amplification. The onetaq reaction buffers and high gc enhancer have been formulated for robust yields with minimal optimization, regardless of a templates gc content. Taq dna polymerase is a thermostable enzyme that synthesizes dna from singlestranded templates in the presence of dntps and a primer.
A protocol for gotaq flexi dna polymerase which provides robust amplification equal to and in some cases superior to conventional 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. 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. 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. Data of expression and purification of recombinant taq dna. Common routes for contamination include using the same pipette to set up a reaction and load. Always check your primer design before constructing or ordering primers. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. 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. The polymerase chain reaction pcr is the cardinal laboratory technology of molecular biology. Using the modified cycling protocol described here, herculase ii fusion dna polymerase also provides. 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. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification. If necessary, the reaction mixture can be overlaid with mineral oil or paraffin melting temperature 5060c of special pcr grade.
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. Pcr protocol for taq dna polymerase with standard taq buffer. Titanium taq pcr kit protocolataglance pt33042 please read the user manual before using this protocolataglance. 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. Taq polymerase, being thermostable, proved ideal for pcr. It has a 53 dna polymerase activity and a 53 exonuclease activity. Cycling conditions amplification parameters depend greatly on the template, primers and amplification apparatus. The polymerase chain reaction pcr was developed by chemist kary mullis in the 1980s, as a means to make many copies of dna fragments. Find additional protocols for other polymerases or advanced pcr techniques in the protocols section of our pcr technologies guide. Moreover, the special enzyme formulation and optimized buffer system ensure robust performance when amplifying difficult and gcrich targets.
Pcr protocol for taq dna polymerase with standard taq buffer m0273 protocols. Taq dna polymerase is a highly thermostable dna polymerase of the thermophilic bacterium thermus aquaticus. Hot start pcr protocol taq dna polymerase is withheld until the reaction temperature is at 80c, to ensure high specificity. The polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. The 5x gotaq green and colorless reaction buffers supplied with gotaq dna polymerase contain mgcl 2 at a concentration of 7.
This product is supplied with 10x reaction buffer containing 15 mm magnesium chloride. Pcr protocol for onetaq dna polymerase m0480 protocols. Onehalf of the total reaction volume is usually sufficient. Therefore, during the extension step, allow approximately 2 minutes for every 1kb to be amplified minimum extension time of 1 minute. Contamination can be a major problem, unless you are careful. Taq dna polymerase is a very important enzyme for molecular biological studies such as dna amplification and dna sequencing. Themagnesium concentrationdoesnot need to be adjusted. Pcr master mix certificate of analysis 9pim750 promega. 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. Therefore, taq dna polymerase can efficiently synthesize dna under the heatintensive conditions of the pcr reaction. The following guidelines are provided to ensure successful pcr using nebs taq dna polymerase. Pfu dna polymerase is another thermostable enzyme that has an optimal elongation temperature of 75 c. 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. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols.
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. Herculase ii fusion dna polymerase technical services us and. Learn more about standard pcr, including what it is, on our pcr basics page. Pcr protocol for taq dna polymerase with standard taq buffer m0273. This abbreviated protocol is provided for your convenience, but is not intended for firsttime users.
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. Protocol to prepare several parallel reactions and to minimize the. The extension rate of pfudna polymerase is lower than that of taq dna polymerase. Scientists realized that thermostable heatstable dna polymerases would be needed for pcr to work efficiently. Taq dna polymerase is an enzyme widely used in pcr. Taq dna polymerase, from thermus aquaticus recombinant.
L reaction and can be used as a starting point for reaction optimization. Taq dna polymerase is a thermostable enzyme derived from. 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. 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. Taq dna polymerase is a thermostable dna polymerase isolated from an e.
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. Pcr the polymerase chain reaction pcr is a powerful and sensitive technique for dna amplification 1. The key is the taq polymerase that was purified from the thermophile thermus aquaticus. A protocol for the use of gotaq dna polymerase with 5x green gotaq reaction buffer and 5x colorless gotaq reaction buffer. For multiple reactions, prepare a master mix of components common to all. Herculase ii fusion dna polymerase provides accuracy comparable to pfu dna polymerase. This dnarna heteroduplex acts as a priming site for the dna polymerase that binds and produces the complementary strand. The key to the polymerase chain reaction was first discovered in1976. Product information polymer taq dna polymerase of reactions. Allows optimization of mgcl 2 concentration and facilitates directtogel analysis of pcr products. Pluthero 1993 rapid purification of highactivity taq dna polymerase. Platinum taq dna polymerase is a recombinant taq polymerase complexed with a proprietary antibody that blocks the polymerase activity at ambient temperatures. This is a basic pcr protocol using taq dna polymerase. Purification of taq dna polymerase for 1 liter culture modified from the protocol presented in f.
These are available online in convenient and compact pdf. Datasheet for taq dna polymerase with standard taq buffer neb. Taq dna polymerase is the most common polymerase used for pcr. Pcr protocol for taq dna polymerase with standard taq buffer m0273 overview. The following protocol is a general guideline and starting point for pcr amplification, however, as with all polymerases, optimal reaction conditions for incubation. Taq buffer with kcl and taq buffer with nh 4 2 so 4. The enzyme consists of a single polypeptide with a molecular weight of 94 kda. Product information polymer taq dna polymerase of reactions plus one extra. 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. The structure of taq polymerase determined here at 2. The supplied 5x green and colorless flexi reaction buffers do not contain magnesium. Use platinum taq dna polymerase for the amplification of dna from complex genomic, viral, and plasmid templates, as well as in rtpcr. Titanium taq can be used in a wide variety of pcr applications, and the constraints on.
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