DNA / PCR

Klenow-fragment of Taq DNA polymerase (KTQ) 2x PCR Master Mix

 

is the perfect choice for standard PCRs and short amplicons. KTQ DNA polymerase supersedes the thermostability of Taq DNA polymerase, the relative mutation rate is twofold lower, but is less processive than Taq DNA polymerase. The KTQ DNA polymerase has no exonuclease or endonuclease activity.
KTQ DNA polymerase provides robust PCR performance for a wide range of PCR applications. The pre-ready 2x mix ensures reproducible results, significantly reduces set-up times and the risk of pipetting mistakes. It can also be used for realtime cycling, when adding a suitable amount of a respective realtime dye.

KTQ 2x PCR Master Mix is a ready to use reaction mix. It contains all components necessary for a successful and reliable PCR or primer extension reaction in all standard PCR cyclers. Only primers and template need to be added. 

 

ArticleProductSizePrice
#5001 SKTQ 2x PCR Master Mix100 reactions á 50 µl, 2x 1.25 ml € 45,-
#5001 MKTQ 2x PCR Master Mix500 reactions á 50 µl, 10x 1.25 ml € 200,-

Bulk offer: Should you require larger amounts (>1000 reactions), significant discounts are available. Please contact us to receive an individual quotation.

 

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Example Results

Example Results

KTQ: Extreme thermostability2014_05_13

Comparison of  thermostability of Taq and KTQ DNA polymerase.

A fragment (92 bp) of the human actin was amplified from 10 ng of a human genomic DNA extract either with Taq or KTQ DNA polymerase with incubation at 95 – 97° C for one hour. PCR products were subsequently analysed on a 2.5% agarose gel.

 

Reaction Setup

Reaction Setup

ComponentVolumeFinal concentration
KTQ 2x PCR Master Mix25 µl1x
Primer forward (10 µM)*1 µl0.2 µM (0.05-1 µM)
Primer reserve(10 µM)*1 µl0.2 µM (0.05-1 µM)
Template/Sample extract**x µl 
Nuclease-free waterup to 50µl total reaction volume 

Keep all components on ice.
Spin down and mix all solutions carefully before use.
* Primers should ideally habe a GC-content of 40-60%
**Suggested template concentration should be about 1 ng - 1 µl (gnomic DNA) or 1 ng - 1 pg (plasmid/viral DNA).

Applications

Applications

  • Standard PCR
  • Realtime PCR
  • Primer extension reaction
  • TA cloning
  • 3`A-tailing of blunt ends
  • Screening / Hight-throughput PCRs

Typical PCR protocol

Typical PCR protocol

StepTemperatureTime
Initial denaturation95°C2 min
Denaturation95°C15 sec
Annealing*54-72°C30 sec     (25-40 cycles)
Extension72 °C2 min / 1000 bp
Hold<10°Chold

*Typically, the annealing temperature is about 3-5°C below the calculated melting temperature of the primers used. 

 

Quality Control

PCR activity: KTQ DNA polymerase was tested for successful PCR performance. A 92 bp fragment (beta-actin gene) was amplified from human genomic DNA and analyzed by agarose gel electrophoresis.

DNA polymerase activity: KTQ DNA polymerase activity has been monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and a DNA primer.
Enzyme-concentration has been determined by protein-specific staining. Please inquire more information at info@mypols.de for the lot-specific concentration.
No contamination has been detected in standard test reactions.

 

Storage

This product is shipped on cool packs. Please store the product upon arrival at -20°C. Minimize the number of freeze-thaw cycles by storing in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4°C.

 

Composition / Content of the Product

KTQ 2x PCR Master Mix is containing all the components necessary for PCR, including wild-type Klenow-fragment of Taq DNA polymerase (KTQ) and an optimized buffer including ultrapure dNTPs.

 

KTQ-teaser

 

img-faqsFAQ`s

Where can I read more about the KTQ DNA polymerase?

 

Have a look under following references for example:

The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion.
Gene 1992; 112: 29–35. W. M. Barnes.

 

Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. 
EMBO J. 1998; 17: 7514–7525. Y. Li, S. Korolev and G. Waksman.

How can I optimize the PCR reaction conditions?

1. The annealing temperature can usually be optimized. Try a temperature gradient and determine the best annealing temperature, which yields in the cleanest product.

2. Add a gradual amount of betaine 0-1 M or DMSO 0-7.5% to the reaction mix and select for the cleanest product and the highest yield.

3. Try to shorten the extension and annealing time. Too long and too many cycles may lead to over-amplification and side-products.

 


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