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Platinum® Pfx DNA Polymerase
Overview
Platinum® Pfx DNA Polymerase is a proprietary enzyme preparation containing recombinant DNA polymerase from Pyrococcus sp. Platinum® Pfx DNA Polymerase possesses a proofreading
Platinum® Pfx DNA Polymerase
Overview
Platinum® Pfx DNA Polymerase is a proprietary enzyme preparation containing recombinant DNA polymerase from Pyrococcus sp. Platinum® Pfx DNA Polymerase possesses a proofreading
Why doesn't Pfx DNA Polymerase yield the same quantitiy of PCR fragment as other thermostable proofreading polymerases?
Product FAQ
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.
Answer Id: 3051
What is the fidelity of Platinum® Pfx DNA polymerase (please quantitate)?
Product FAQ
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
Answer Id: 3026
How should I adjust the Platinum® Pfx DNA Polymerase protocol if I am trying to generate an amplicon greater than 2 kb, or if I am starting with long PCR primers?
Product FAQ
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.
Answer Id: 3053
Why did I get a lower yield of long PCR product with Platinum® Pfx than with a polymerase mix such as Elongase® enzyme?
Product FAQ
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.
Answer Id: 3052
AccuPrime™ Pfx DNA Polymerase | Life Technologies
Experiment Protocol
Can I use Platinum® Taq DNA Polymerase High Fidelity or Platinum® Pfx DNA Polymerase for site-directed mutagenesis?
Product FAQ
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.
Answer Id: 3887
Platinum® Taq DNA Polymerase
Overview
Platinum® Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that inhibits polymerase activity at ambient temperatures.
Can I use a proofreading enzyme such as Platinum®, Pfx™, Vent, or Pfu with TOPO® TA Cloning® kit?
Product FAQ
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.
Answer Id: 3333
Do Elongase® and Platinum® Taq High Fidelity enzymes leave a 3'-A overhang on the PCR product for subsequent cloning into a TOPO® TA Cloning® or original TA vectors? What about Platinum® Pfx polymerase?
Product FAQ
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.
Answer Id: 3064
Platinum Pfx DNA Polymerase
Manual / Product Insert
Platinum Pfx DNA Polymerase
Product Literature
- Download
- Version: 20 August 2002
Platinum Pfx DNA Polymerase 1
Product Literature
- Download
- Version: 20 August 2002
Platinum Pfx DNA Polymerase 2
Product Literature
- Download
- Version: 20 August 2002
