( ...)Efficient and Reliable PCR Setup Using Eppendorf MasterMix

Christian Rohrer and Lars-Erik Peter
Christian Rohrer, Eppendorf AG, Germany
Lars-Erik Peters, Eppendorf 5Prime, USA

Introduction

The reliability of a PCR* experiment is dependent on its sensitivity, specificity and reproducibility. An essential prerequisite for a successful PCR is efficient sample preparation, as well as thermal cyclers with excellent temperature precision and block homogeneity.

Preparing a PCR reaction using different reaction components is afforded a particularly crucial role since the precision of the reagent concentrations from tube to tube, as well as from one experiment to the next, must be guaranteed. When a PCR preparation is being carried out, a series of comprehensive pipetting steps are necessary. It is advisable to minimize the number of pipetting steps as every additional step is accompanied by the risk of reduced precision and increased contamination.

Only constant concentration of the reaction components from one preparation to the next can guarantee the best possible reproducibility of the individual reactions. Ready-to-use MasterMix eliminates the need for time-consuming pipetting steps, since only primer and template need to be added to 2.5 x MasterMix. This not only minimizes the risk of errors, but it also optimizes precision, as well as saves a great deal of time.

In addition to reagents such as Taq polymerase, nucleotides and a buffer system, MasterMix contains special stabilizers, which ensure high performance in the PCR and excellent stability of the reaction components.

Objective of the Test

Eppendorf MasterMix was tested under various conditions. The objective was to test the reproducibility of the results and to ascertain the stability of the MasterMix at different storage and application temperatures. Tests were carried out on different types of applications and different individual applications.

Materials and Methods

Experiment 1:
Reproducibility of parallel PCR preparations tested using Eppendorf MasterMix
In 56 parallel preparations, a 400-bp fragment from the gene of the Taq polymerase (60% GC content) was amplified. 15 l template/primer mix was added to 10 l of 2.5 x MasterMix (per preparation: 1.5 mM MgCl2, 200 M each of dNTP, 1.25 U Taq; 200 pg pTTQ18 plasmid DNA, 20 pmol per primer). The reaction took place in a 0.2 ml PCR tube with the following PCR program on Mastercycler gradient** from Eppendorf.

94C 3 minute initial denaturation
30 cycles:
94C 15 seconds denaturation
59C 10 seconds annealing
72C 40 seconds extension

The fragments were separated by electrophoresis in a 1.2% agarose gel in TAE buffer at 110 V.

Experiment 2:
Stability test for PCR MasterMix: Using Eppendorf MasterMix after storage under different condition temperatures in three different PCR systems.
Following incubation for 48 hours each at 20C, 28C, 2028C or 37C, Eppendorf MasterMix was tested in three different PCR systems. According to the system used, a 360-bp fragment from the human gene apolipoprotein B (Exon 26), a 547-bp fragment with the coding sequence of the pyrophosphatase gene of Thermo thermophilus (70% GC content) and a 10-kb fragment from the genome of the phage lambda was amplified. The target DNA used was human genomic DNA, T.th genomic DNA or lambda DNA. The PCR reaction volumes were each 50 l. 30 l template/primer mix was added to 20 l of 2.5 x MasterMix (per preparation: 1.5 mM MgCl2, 200 M each of dNTP, 1.25 U Taq; 20 ng human genomic DNA, 10 ng T. thermophilus HB8 genomic DNA or 25 ng lambda DNA; 20 pmol per primer). The reaction took place in a 0.2 ml PCR tube with the following PCR programs on the Mastercycler gradient:

Human genomic DNA
94C 3 minute initial denaturation
35 cycles:
94C 20 seconds denaturation
52C 10 seconds annealing
72C 25 seconds extension

T.th genomic DNA
95C 3 minute initial denaturation
35 cycles:
94C 20 seconds denaturation
55C 10 seconds annealing
72C 40 seconds extension

Lambda DNA
93C 3 minute initial denaturation
20 cycles:
93C 15 seconds denaturation
62C 30 seconds annealing
68C 12 seconds extension

The fragments were separated by electrophoresis in a 1.2% agarose gel in TAE buffer at 70 V.

Experiment 3:
Application spectrum of Eppendorf MasterMix regarding target fragment length and template
The area of use of MasterMix was tested by amplifying fragments of different lengths (160 bp10 kb). Four different templates were used: human genomic DNA, T. thermophilus HB8 genomic DNA, plasmid DNA (pTTQ Taq) and lambda DNA.

The PCR reaction volumes were each 50 l. 30 l template/primer mix was added to 20 l of 2.5 x MasterMix (per preparation: 1.5 mM MgCl2, 200 M each of dNTP, 1.25 U Taq; 20 ng human genomic DNA, 10 ng T. thermophilus HB8 genomic DNA or 25 ng lambda DNA; 20 pmol per primer). The reaction took place in a 0.2 ml PCR tube with the following PCR program on the Mastercycler gradient:

Human genomic DNA
94C 3 minute initial denaturation
35 cycles:
94C 20 seconds denaturation
52C 10 seconds annealing
72C 25 seconds extension

T.th genomic DNA
95C 3 minute initial denaturation
35 cycles:
94C 20 seconds denaturation
55C 10 seconds annealing
72C 40 seconds extension

Lambda DNA
93C 3 minute initial denaturation
20 cycles:
93C 15 seconds denaturation
62C 30 seconds annealing
68C 12 seconds extension

Plasmid DNA (pTTQ Taq)
94C 3 minute initial denaturation
30 cycles:
94C 15 seconds denaturation
59C 10 seconds annealing
72C 40 seconds extension

The fragments were separated by electrophoresis in a 1.2% agarose gel in TAE buffer at 70 V.

Results

Experiment 1:
As shown in Fig. 1, the same product was amplified in all PCR preparations, with the bands all showing the same degree of intensity. MasterMix is an easy-to-handle, timesaving method of guaranteeing optimal reproducibility from one experiment to the next.


Fig. 1: Reproducibility of parallel PCR preparations with MasterMix A 400-bp fragment from the gene of Taq DNA polymerase was amplified using MasterMix.

Experiment 2:
As shown in Fig. 2, pre-incubating MasterMix for 48 hours at a deep-freeze temperature, refrigerator temperature, room temperature or thermostat temperature (37C) had no adverse effect on the subsequent performance of the reaction components in the PCR. MasterMix is characterized by maximum thermostability and, at the same time, high processivity and specificity.



Fig. 2: Stability test for Eppendorf MasterMix: Using MasterMix after storage at four different temperatures in three different PCR systems

Lanes 14: A 360-bp fragment of the human apolipoprotein B was amplified from genomic DNA.
Lanes 58: A 547-bp fragment of the pyrophosphatase from Thermus thermophilus HB8 was amplified from genomic DNA.
Lanes 912: A 10-kb fragment from lambda DNA was amplified.
Lane 13: MBI Fermentas GeneRuler (100 bp10 kb)

Experiment 3:
MasterMix shows high performance levels across the entire applications range tested (Fig. 3). Fragments of different sizes can be amplified with high yields and high specificity, irrespective of the template used.



Fig. 3: Applications spectrum of Eppendorf MasterMix with regard to target fragment length and template

Lane 1: 160-bp fragment from exon 26 of the human gene of the apolipoprotein B, (genomic DNA)
Lane 2: 360-bp fragment from exon 26 of the human gene of the apolipoprotein B, (genomic DNA)
Lane 3: 400-bp fragment from the gene of Taq DNA polymerase (60% GC), (plasmid DNA)
Lane 4: 547-bp fragment from the coding sequence of the pyrophosphatase gene Thermus thermophilus (70% GC), (genomic DNA)
Lane 5: 10-kb fragment from the genome of phages lambda DNA)
Lane 6: MBI Fermentas GeneRuler (100 bp10 kb)

Conclusion

Using the prepackaged MasterMix with the PCR is characterized by safe and rapid handling: simply add primer and template, and the PCR is ready to go. Reducing pipetting steps not only optimizes the time required but also reduces the risk of contamination and minimizes pipetting errors. Since MasterMix can be stored at 4C, the time-consuming process of thawing reagents is no longer necessary. Special stabilizers make MasterMix extremely thermostable, meaning that temperatures of 20C, room temperature or even 37C have no effect on its performance (see Experiment 2). In comparison to conventional PCR reagents, Eppendorf MasterMix is thus considerably easier to handle in the lab. MasterMix guarantees excellent reproducibility of PCR results (see Experiment 1), with pipetting errors or incorrect preparation of the reagent virtually ruled out.

The consistently high quality of each batch of reaction components, as well as their concentration is subjected to thorough tests during manufacture. MasterMix was successfully tested with a variety of different templates (see Experiment 3), with a wide spectrum of fragments being amplified (160 bp10 kb). Specific, high-yield amplification of very long fragments (10 kb) is also possible using MasterMix without the need to purchase special additional long-range enzymes. Safe to use and with optimized time requirements, MasterMix is ideal for high-throughput laboratories. In addition, its vast applications spectrum and high stability make it the ideal tool for research laboratories that work with different templates and amplicon lengths on a daily basis.

*Practice of the patented polymerase chain reaction (PCR) process requires a license.

** The Eppendorf Thermal Cycler is an Authorized Thermal Cycler and may be used with PCR licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Mastercycler gradient (U.S. Pat. 6,210,958)

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