The FastStart PCR Master is a convenient and ideal tool for direct colony PCR. Intact bacteria can be analyzed directly without prior template purification, FastStart PCR Master is therefore the tool of choice for rapid screening of cloning experiments. In this paper, we describe the use of the FastStart PCR Master for the amplification and cloning of mRNA including direct colony screening. An overview of the workflow is presented in Figure 1.
The Roche Applied Science FastStart PCR Master is a fast and convenient ready-to-use, 2x concentrated master mix that contains all reagents (except PCR primers and template) needed for running hot-start PCR on thermal block cycler instruments: FastStart Taq DNA Polymerase, magnesium chloride, double-concentrated reaction buffer, stabilizers and nucleotides (dATP, dCTP, dGTP, dTTP, 0.4mM each). Hot-start protocols have been shown to significantly improve the specificity, sensitivity, and yield of PCR.
Materials and Methods
Total RNA was obtained from K562 cells using the High Pure RNA Isolation Kit as described in the package insert. An aliquot of the isolated RNA was reverse transcribed to cDNA with the Transcriptor First Strand cDNA Synthesis Kit. The cDNA synthesis was performed in two-step RT-PCR as described in the package insert using random hexamers and a conventional thermocycler.
The FastStart PCR Master Mix was used in comparison with the Pwo SuperYield DNA Polymerase for subsequent amplification of the cDNA. Pwo SuperYield DNA Polymerase (also available as the convenient Pwo Master) is a proofreading polymerase and shows 18-times higher fidelity compared with FastStart Taq DNA Polymerase and FastStart PCR Master. The desired target of the amplification was a 380-bp fragment of the human interleukin gene (IL-2). Gene-specific primers (5´CTC ACCAGGATGCTCAC, 3´GTAGCAAACCATACATT) were used for PCR. The amplification reaction was performed according to the package insert of each polymerase. Each reaction was cycled according to the following parameters: The thermocycling protocol consisted of an initial incubation step at 94°C for 2 minutes. The following cycling profile was repeated for 35 PCR cycles: 30seconds at 94°C, 30 seconds at 55°C, 45 seconds at 68°C. The extension step was extended by 5 seconds for the last 25 cycles. The final extension step at 68°C was held for 7 minutes.
An aliquot of each reaction was analyzed on a 1% agarose gel (Agarose MP) stained with 1 µg/ml ethidium bromide and compared with DNA Molecular Weight Marker VIII to verify the success of the PCR. The PCR product was purified with the High Pure PCR Product Purification Kit as described in the package insert. Subsequently, the PCR products generated with the FastStart PCR Master were polished with T4 DNA Polymerase to generate blunt-ended PCR fragments as follows: 1 U of T4 DNA Polymerase was incubated with the 300 ng of the PCR product, T4 DNA Polymerase reaction buffer and dNTPs for 30 minutes at 37°C. Subsequently, the polished PCR product was purified with the High Pure PCR Product Purification Kit.
Pwo SuperYield DNA Polymerase does not exhibit terminal transferase activity and generates blunt-ended PCR fragments. Therefore, polishing was not required for fragments generated with Pwo SuperYield DNA Polymerase
The amplification product was cloned into pCAPs cloning vector using the PCR Cloning Kit (blunt-end). Vector restriction, ligation, and control reaction were performed according to the package insert of the kit. Each ligation reaction of the PCR products generated with the two different polymerases was transformed as indicated in the package insert of the PCR Cloning Kit (blunt-end) in competent E. coli DH5a cells. Different amounts (50– 100µl) were plated on selective LB plates supplemented with 100µg/ml Ampicillin. The plates were incubated for 16–18hours at 37°C. Subsequently, ten clones of each approach were randomly picked with sterile toothpicks and heated in 50 µl H2O for 5 minutes at 95°C. Following centrifugation at 13,000 rpm for 1 minute, direct colony PCR of the supernatant was performed with the FastStart PCR Master Mix and gene-specific primers to identify positive clones. PCR cycling parameters were as follows: initial denaturation at 94°C for 2 minutes; 10cycles at 94°C for 30 seconds, 55°C for 30 seconds and 68°C for 45 seconds; 20cycles at 94°C for 30 seconds, 55°C for 30 seconds and 68°C for 45 seconds with a extension time of 5 seconds per cycle; final extension at 68°C for 7 minutes. cDNA amplification was analyzed by agarose gel electrophoresis (Figure 2).
Results and Discussion
PCR for template amplification
Agarose gel analysis showed that the polymerases consistently amplified a 380-bp fragment (Figure 2). FastStart PCR Master and the Pwo SuperYield DNA Polymerase yielded comparable amounts of amplification product.
PCR for colony screening
The colonies were counted and more than 50 colonies were obtained from each cloning experiment.
FastStart PCR Master was used for direct colony PCR. All 20 randomly selected clones showed an insertion of the 380-bp fragment ligated into the plasmid vector pCAPs (Figures 3, 4). Consequently, the cloning experiment succeeded independently of the PCR system used for template amplification. The FastStart PCR Master is extremely suitable for PCR performed directly on E. coli cells. Furthermore, both amplification products were highly suitable for subsequent cloning.
The convenient ready-to-use FastStart PCR Master mix is an ideal tool for standard amplification using hot-start protocols. In addition, it offers the advantage of usage in direct colony PCR. As demonstrated above, the FastStart PCR Master Mix shows high convenience and allows high-troughput screening of colonies with only one reagent. Colony PCR is a handy technique for simplifying the detection of successful cloned inserts. It enables a highly efficient workflow and replaces the time-consuming conventional clone analysis including plasmid preparation and analysis.
The results clearly demonstrated that the new FastStart PCR Master is a useful, reliable and versatile hot-start tool for both applications: PCR product amplification and direct colony screening. Therefore, the FastStart PCR Master is an ideal tool for fast and convenient PCR applications.
This article was originally published in Biochemica 1/2006, pages 14-15. ©Springer Medizin Verlag 2006.