Invitrogen™ Platinum™ GenoType Tsp DNA Polymerase

Catalog No.	No. of Reactions
11-448-024	400 Reactions
11-448-032	2000 Reactions

Catalog No. 11-448-024 Supplier Invitrogen™ Supplier No. 11448024

Description

Includes

Platinum GenoType TspDNA Polymerase is supplied with 10X PCR Buffer (200mM Tris-HCl (pH 8.4), 500mM KCl), and a vial of 50mM MgCl₂.

Platinum GenoType Tsp DNA Polymerase is designed and qualified specifically for amplification of dinucleotide repeat markers in PCR-based genotyping applications.

Platinum GenoType Tsp DNA Polymerase is designed and qualified specifically for amplification of dinucleotide repeat markers in PCR-based genotyping applications. The enzyme is pre-complexed with a thermolabile inhibitor containing monoclonal antibodies to Tsp DNA polymerase, providing an automatic hot-start method that improves PCR specificity and allows for room temperature reaction assembly.

Platinum GenoType Tsp DNA Polymerase features

• Minimal non-templated nucleotide addition to PCR products
• Lacks both 5' and 3' exonuclease activity
• Amplifies fragments up to 500 bp
• Room temperature reaction assembly

Application

Amplification of dinucleotide repeat loci in applications in which elimination of non-templated nucleotide addition is desirable.

Unit definition

One unit of Platinum GenoType Tsp DNA Polymerase has been functionally determined to be equivalent to one unit of Taq DNA Polymerase in amplification of dinucleotide repeats using standard Taq DNA polymerase reaction conditions. One Tsp DNA polymerase unit approximates 2.5 activity units. An activity unit incorporates 10 nmoles of deoxyribonucleotide into acid-precipitable material in 30 min. at 74°C under optimized reaction conditions.

Order Info

Approved for shipment on Wet or Dry Ice

Specifications

• Content And Storage: • Platinum GenoType Tsp DNA Polymerase (50 μL at 5 Tsp U/μL)
  • 10X PCR Buffer (1.25 mL)
  • 50 mM MgCl₂ (1 mL)
  
  Store at -20°C. Guaranteed stable for 6 months when properly stored.
• GC-Rich PCR Performance: Low
• Polymerase: Tsp Genotyping Polymerase
• Reaction Speed: Standard
• Quantity: 250 units
• Shipping Condition: Wet or Dry Ice
• For Use With (Application): Hot-start PCR
• Fidelity (vs. Taq): 1X
• Hot Start: Built-In Hot Start
• No. of Reactions: 400 Reactions
• Overhang: Blunt
• Reaction Format: Separate Components
• Size (Final Product): 500 bp or less
• Volume: 50 μL

Frequently Asked Questions (FAQs)

What is the difference between Platinum technology and AccuPrime technology?

With Platinum technology, anti-DNA polymerase antibodies bind to the enzyme until the denaturing step at 94 degrees C, when the antibodies degrade. The polymerase is now active and primer extension can occur. AccuPrime Taq combines Platinum Taq (Taq + Platinum antibodies) with proprietary thermostable AccuPrime accessory proteins. The 10X reaction buffer contains the accessory proteins which enhance specific primer-template hybridization during each cycle of PCR.

Is there anything to prevent AmpliTaq Gold DNA polymerase from extending from the 3’ end of a TaqMan probe in a 5’ nuclease assay?

Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.

How does AmpliTaq Gold DNA Polymerase differ from AmpliTaq DNA Polymerase?

AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp 10X PCR Buffer I and/or GeneAmp 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

Does AmpliTaq Gold DNA Polymerase contain exonuclease (proofreading) activity?

No, AmpliTaq Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.

Does the fidelity of AmpliTaq DNA Polymerase change in the presence of base analogs?

The fidelity of this PCR enzyme is affected in two ways. First, AmpliTaq DNA Polymerase typically binds to and incorporates base analogs less efficiently than conventional dNTPs, which means that polymerase activity is lower in reactions that contain base analogs. Second, the analog may pair with more than one conventional complementary template base, so the analog may be incorporated at an increased level compared to conventional dNTPs. For the best fidelity, we recommend that base analogs are included at low concentrations in the reaction.

What is the expected half life of AmpliTaq DNA Polymerase at 95 degrees C?

The half-life of AmpliTaq DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t_1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.

What is "Hot-start" PCR?

Hot-start is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. The major obstacle to obtaining highly sensitive and specific amplifications appears to be competing side reactions such as the amplification of non-target sequences (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Mispriming can occur any time all components necessary for amplification are present at permissive temperatures (below optimal annealing temperature) such as during reaction set up. A hot start can be performed either manually or can be automated utilizing AmpliTaq Gold DNA Polymerase.

In the manual hot-start technique a key component necessary for amplification, such as the enzyme, is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. Once this temperature is reached, the missing component is added and the PCR amplification is allowed to proceed. Because a key component was withheld from the reaction at permissive temperatures, competing side reactions are minimized and specific amplification occurs.

AmpliTaq Gold DNA Polymerase facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase. Once activated, AmpliTaq Gold DNA Polymerase performs just as AmpliTaq DNA Polymerase does. Since it is provided in its inactive form, it can be added to a reaction without the fear of pre-PCR misprimed primers being extended. Once all of the components for amplification have been added to a tube, the reaction is heated to 95C for 5 - 10 minutes. This incubation activates the enzyme and allows the reaction to proceed normally.

How much MgCl2 should be added to the PCR amplification when using AmpliTaq DNA Polymerase or AmpliTaq Gold DNA Polymerase?

The standard starting point is a final concentration of 1.5 mM magnesium ion. Since each molecule of dNTP (total 0.8 mM per reaction at 200 µM each) binds a magnesium ion, 0.8 mM magnesium ions are unavailable for AmpliTaq DNA Polymerase to use; hence, 0.7 mM free magnesium ions will be available as a cofactor for Taq's polymerization activity. It is important to note that there are other substrates in PCR amplifications that can also bind free magnesium (such as primers and template) therefore, the magnesium ion concentration should be titrated in order to find the optimum concentration for each reaction.

How much AmpliTaq DNA Polymerase is used in a PCR amplification?

Most PCR amplifications use 2.5 units of AmpliTaq DNA polymerase per 100 µL reaction. A 25 µL reaction would use about 0.6 units. However, the optimal unit concentration per reaction should be empirically determined, and often the less is better rule applies. Using too much AmpliTaq may result in non-specific amplification.

When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?

The concentration of dNTPs in a standard PCR amplification is 200 µM each, for a total of 800 µM. This total dNTP amount corresponds to 39 µg of dNTPs. This is a huge excess and, when generating long PCR fragments, is not a limiting factor during the PCR amplification, as the amount of target DNA generated is generally no more than 1 µg. More importantly, the reaction condition variables need to be monitored more closely in order for successful long PCR amplification to occur.

What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?

Successful amplification of long PCR targets is dependent on variables such as sufficient extension time during the PCR amplification, cosolvent addition, pH of the reaction buffer, salt concentration, primer design, use of a hot start, DNA sample integrity, and the enzyme's proofreading and polymerase activities. A few examples of our long PCR enzymes include our Elonagase enzyme mix that can be used for amplicons up to 30kb (blend of Taq and proofreading enzyme) or our Phire Hot Start II enzyme mix that can be used for amplicons up to 20 kb (Taq polymerase). Read more here: https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/long-fragment-pcr.html

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov

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