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View additional product information for Platinum™ Pfx DNA Polymerase - FAQs (11708013)
19 product FAQs found
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.
Yes. dUTP inhibits the activity of archaebacterial polymerases such as Pfx DNA Polymerase.
Yes, both enzymes are suitable for site-directed mutagenesis. However, Platinum Pfx Polymerase may not be compatible with the site-directed mutatgensis system from Stratagene because the required concentration of template is not sufficient.
Proofreading enzymes possess 3'-5' exonuclease activity (Gene 112:29 (1992)) which removes 3'-A overhangs necessary for TA and TOPO TA Cloning kit. Since the PCR products are mostly blunt-ended, the use of these PCR products in TA cloning yields very low cloning efficiencies. We have developed a simple protocol for adding the 3' A overhang to these PCR products so that they can be used in the TA cloning reaction.
Before starting, you will need the following items:
-Taq polymerase
-A heat block equilibrated to 72 degrees C
-Phenol-chloroform
-3M sodium acetate
-100% ethanol
-80% ethanol
-TE buffer
Procedure
-After amplification with Vent, Pfx, or other proofreading polymerases, place samples on ice and add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to change the buffer or remove the proofreading polymerase. A sufficient number of PCR products will retain the 3'-A overhangs.
-Incubate at 72 degrees C for 8-10 min (do not cycle).
-Place on ice and use immediately in the cloning reaction.
-If you need to store the samples overnight, extract the sample immediately with an equal volume of phenol:chloroform. Extraction with phenol-chloroform removes all of the polymerase. Precipitate the DNA by adding 1/10 volume of 3M sodium acetate and 2X volume of 100% ethanol. Keep at -20 degrees C for 20 min or -80 degrees C for 10-15 min. Centrifuge at maximum speed for 5 min at room temperature to pellet the DNA. Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air dry. Resuspend the pellet in TE buffer to the starting volume of the PCR amplification reaction. The PCR amplification product is now ready for ligation into the TA cloning or TOPO TA Cloning vector.
Note: if your amplification has produced more than one PCR product, you may wish to gel-purify the correct fragment after amplification with Pfu or Vent. After purification, add Taq polymerase buffer, dATP, and 0.5 unit of Taq polymerase and incubate 10-15 min at 72 degrees C. Proceed directly to the cloning reaction.
Elongase and Platinum Taq High Fidelity polymerase mixes do leave 3' A overhangs on a portion of the PCR products, however, the cloning efficiency is greatly reduced from that obtained with Taq polymerase alone. Platinum Pfx polymerase does not leave 3' A overhangs. Therefore, with all proofreading enzymes or enzyme mixes that contain proofreading polymerases, we recommend that you treat the PCR product with Taq at the end of the PCR reaction, prior to TA cloning. To do this, add 1 U of Taq to a 50 µL reaction and incubate at 68-72 degrees C for 15 min. Phenol extract and ethanol precipitate the product before TA cloning.
Additional notes: The cloning efficiency decreases with increasing size of PCR products. For larger PCR fragments, we recommend that you gel-purify the PCR product and screen several clones. PCR primers should be designed with a 5' G, since Taq leaves a 3' A overhang preferentially on DNA ending in C.
Reference: Hu (1993) DNA and Cell Biology 12:763.
TA Cloning reference: Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A., and Smith, L.M. (1991) BioTechnology 9, 657.
To generate amplicons greater than 2 kb, use 2.5 units of Platinum Pfx polymerase (instead of 1 unit), decrease the extension temperature to 68 degrees C, and increase the extension time to 1 kb/minute.
If long PCR primers are used with Platinum Pfx polymerase, increase the magnesium concentration in the reaction to 1.5 mM.
While enzyme mixes offer improved fidelity over Taq DNA Polymerase alone, Platinum Pfx DNA Polymerase provides much higher fidelity since it is a proofreading polymerase exclusively. If yield is more important than fidelity, then an enzyme mix such as Platinum Taq High Fidelity or Elongase enzyme mix is a better choice of enzyme (since they contain a significant amount of Taq in addition to the proofreading polymerase). Platinum Pfx does give high yields relative to other proofreaders, but may not give as great a yield as the enzyme mixes.
Generally, it should. However, the 10X Pfx Amplification buffer is optimized to work with Platinum Pfx DNA Polymerase, so it is important Pfx is not used with buffers meant for other polymerases. Additionally, since the magnesium concentration is lower in the Pfx buffer, the primer annealing temperature may need to be lowered.
The rpsL fidelity assay was used to generate this data. Briefly, a plasmid containing an AmpR and SmS gene was amplified by PCR and religated. Transformations were plated on Amp plates and Amp/Sm plates. The mutant frequency = rpsL mutant colonies/total colonies x 100.
Results from this assay:
Taq: 4.8/100,000
Platinum Pfx: 1/1,000,000
Pfu were 1.5/1,000,000
Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.
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).
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.
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.
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 t1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.
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.
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.
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.
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.
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