Navigation Links
The use of TaqMaster PCR Enhancer

Guy Adami, Department of Oral Medicine, University of Illinois in Chicago, USA
Vincent Prezioso, Eppendorf Scientific, Inc., Westbury, NY, USA


Transgenic mice carry a fragment of foreign DNA that has been incorporated into their genome. The most common method of introducing recombinant DNA into the mouse genome is by microinjection of the DNA into the pronuclei of one-cell stage mouse embryos.

A fraction of these embryos develop into newborn mice which have the micro-injected DNA stably integrated in their genome. A second strategy for generating transgenic mice involves genetic manipulation of ES cells.

After electroporation with a carefully designed construct, the stem cells are screened for the integration of new DNA through homologous recombination.

ES cells that are positive for a homologous recombination event are then transplanted into blastocyst stage embryos in order to generate chimeric mice. In most cases, gene targeting is used to introduce mutations into specific genes in the mouse genome.

The transgenic DNA is typically transcribed and translated in a tissue-specific and stage-specific pattern that is determined by regulatory sequences present in the transgenic DNA. Consequently, transgenic mice represent an experimental system in which it is possible to study the consequences and/or pattern of expression (or lack thereof) of the new genetic information. Incorporated transgenes are often detected by genomic DNA isolation from a tissue sample, followed by PCR* amplification of a region of the inserted gene.

The tissue source most commonly used for genotyping is mouse tail (1), however, ear, toe, hair, and blood samples are also used. Typically, genomic DNA isolation procedures from tail slices involve long incubations (one hour to overnight) at high temperature in the presence of Proteinase K. One of the problems that researchers often see is inconsistency in the amplification of transgenic DNA caused by impurities in the template DNA: These often inhibit PCR reactions causing false negative or inconclusive results. Here, we describe a reliable method of achieving consistent amplification of genomic DNA with impurities using a
novel PCR enhancer.

The Eppendorf MasterTaq Kit contains the TaqMaster PCR enhancer that has been specially developed to improve the amplification of difficult templates by improving the thermostability of the Taq DNA Polymerase. TaqMaster also adsorbs impurities in the template making the Taq less sensitive to exogenous PCR
inhibiting contamination. Impure and GC-rich templates show increased yields and reproducible results.


Six newborn mice were screened for the presence of a TGF alpha transgene. Mouse tail DNA (from last 2 mm of tail) was isolated by Proteinase K digestion
followed by heat inactivation as described (2).

Briefly, 2 mm of tail was placed directly into 200 l DNA Extraction Buffer with Nonionic Detergents (PBND) (3) in a 1.5 ml microcentrifuge tube. Next 2.5 l Proteinase K solution at 20 mg/ml (4) was added to each sample.

The samples were incubated at 55 C with occasional vortexing until tissue w as degraded (~2 hours). Finally, the samples were boiled for 10 minutes (to inactivate the Proteinase K), and centrifuged for 5 minutes. 2 l of this reaction was used per PCR reaction. The primers used in the reaction are specific to the human TGF alpha cDNA and were synthesized as described (5).

The size of the expected amplified product is 182 bp. The PCR was split into two sets of reactions, with and without the TaqMaster, according to the manufacturer's specifications (6). After an initial incubation at 94 C for 5 minutes, 0.2 l was added to each reaction. The PCR was run for 29 cycles under the following conditions:
1. Denature 94 C, 1 minute
2. Hybridization 56 C, 30 seconds
3. Extension 70 C, 40 seconds

Figure 1 shows the results of the experiment. All results were confirmed by phenotypic analysis of adult transgenic animals (7).

Figure 1. The TaqMaster enhancer makes the PCR signal both stronger and much more consistent. This is a 1.5 % agarose gel run with TAE. Lanes: 1 & 14 = 1 kb ladder (Life Technologies), Lanes 2-7 PCR without enhancer (TaqMaster), Lanes 8 -13 with the TaqMaster and the markers again.

The lanes with reactions that do not contain the PCR enhancer show variability and artifacts such as primer-dimers (lanes 2-7). Note especially lanes 3 and 4 where a weak positive signal could lead to ambiguity about the results. In contrast, lanes 8 -13 show extremely consistent results. Positive animals can be clearly identified as those corresponding to lanes 9, 10 and 13.

Note that lanes 9 and 10, which are from the same template as lanes 3 and 4, show robust amplification with no primer-dimers or other artifacts. The other lanes with the TaqMaster also show no primer-dimers and strong signals in the case of positive animals.

This demonstrates that the TaqMaster has the effect of making these reactions more robust and consistent.


Eppendorf's TaqMaster PCR enhancer is ideal for PCR reactions that contain templates with impurities.

(1) Hogan, B. Manipulating the Mouse Embryo 1994, p. 296

(2) Perkin Elmer Cetus: Vol.#2 PEC1989, pp. 1-3

(3) PBND buffer contains:
50 mM KCl
10 mM Tris-HCl (ph 8.3)
2.5 mM MgCl
0.1 mg/ml gelatin
0.45 % v/v Nonidet P40 (NP40)
0.45% v/v Tween 20

(4) Proteinase K: Made fresh for each use! 20 mg Proteinase K is dissolved in 1 ml ddH O @ 4 C. Filter sterilize.

(5) Ji H.J., Zhang Q.Q., Leung B.S. Survey of oncogene and growth factor/receptor gene expression in cancer cells by intron-differential RNA/PCR. 1990 Jul 31; 170 (2): pp. 569-575

(6) PCR reaction mix for ten tubes consists of: Rxn. with enhancer:
25 l 10x Buffer
50 l TaqMaster PCR Enhancer
20 l dNTP at 2.5 M
2.5 l Primer 1 at 1 g/l
2.5 l Primer 2 at 1 g/l
130 l H O
+2 l Tail DNA
(out of 200 l total per 2 mm piece of tail)
Rxn. without enhancer:
25 l 10x Buffer
20 l dNTP at 2.5 M
2.5 l Primer 1 at 1 g/l 20 mer
2.5 l Primer 2 at 1 g/l 20 mer
180 l H O
+2 l Tail DNA
(out of 200 l total per 2 mm piece of tail)

(7) Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, Merlino GT. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. 1990 June 15; 61 (6): pp. 1137-1146

Applicational tip: PCR sample preparation



Page: All 1 2 3 4 5

Related biology technology :

1. Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
2. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
Post Your Comments:

(Date:11/27/2015)... (PRWEB) , ... November 27, 2015 , ... ... Program that includes over 2,000 technical presentations offered in symposia, oral sessions, ... and applied spectroscopy, covers a wide range of applications such as, but not ...
(Date:11/26/2015)... November 26, 2015 ... Accutest Research Laboratories, a leading ... Organization (CRO), has formed a strategic ... - Temple Health for joint work ... (Photo: ) , --> ...
(Date:11/25/2015)... , November 26, 2015 ... Global Biobanking Market 2016 - 2020 report analyzes ... maintaining integrity and quality in long-term samples, minimizing ... long-term cost-effectiveness. Automation minimizes manual errors such as ... technical efficiency. Further, it plays a vital role ...
(Date:11/25/2015)... 2 nouvelles études permettent d , identifier ... souches bactériennes retrouvées dans la plaque dentaire des ... Ces recherches  ouvrent une nouvelle voie ... l,un des problèmes de santé les plus fréquemm ... --> 2 nouvelles études permettent d , ...
Breaking Biology Technology:
(Date:11/18/2015)... Nov. 18, 2015  As new scientific discoveries deepen ... and other healthcare providers face challenges in better using ... patients. In addition, as more children continue to survive ... adulthood and old age. John M. Maris, ... Hospital of Philadelphia (CHOP) . --> ...
(Date:11/17/2015)... PARIS , November 17, 2015 ... November 2015.   --> Paris from ... --> DERMALOG, the biometrics innovation leader, has invented the ... and fingerprints on the same scanning surface. Until now two ... fingerprints. Now one scanner can capture both on the same ...
(Date:11/17/2015)... 17, 2015  Vigilant Solutions announces today that Mr. ... of Directors. --> --> ... from the partnership at TPG Capital, one of the ... $140 Billion in revenue.  He founded and led TPG,s ... TPG companies, from 1997 to 2013.  In his first ...
Breaking Biology News(10 mins):