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faststart taq manualTemplate purity and quality are also critical to PCR success. Sequence and primer concentrations also determine overall assay quality. Nucleotides are vital components in amplification reactions and purity and concentration of these reagents significantly influences PCR results. In some cases, additives can enhance PCR efficiency, specificity, and yield. The appropriate cycling parameters contribute to a successful PCR. In addition, refer to the interactive PCR Product Selection Guide linked to this site. Reproduction of any materials from the site is strictly forbidden without permission. Sigma-Aldrich Products are sold exclusively through Sigma-Aldrich, Inc. FastStart Taq DNA Polymerase is an ideal tool for hot start PCR, because the enzyme remains inactive during PCR set-up and prior to the initial denaturation step.Each lot is also tested for the absence of exo- and endonucleases and nicking activity.The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation.Not for use in diagnostic procedures.This product is for research use only. Human and veterinary diagnostic uses under Roche patent claims require a separate license from Roche. All uses other than internal research and human and veterinary diagnostic uses under Roche patent claims require a separate license from Thermo Fisher Scientific. Further information on purchasing licenses from Roche may be obtained by contacting the Licensing Department of Roche Molecular Systems, Inc., 4300 Hacienda Drive, Pleasanton, California 94588, USA or Roche Diagnostics GmbH, Sandhofer Strasse 116, 68305 Mannheim, Germany. Further information on purchasing licenses from Thermo Fisher Scientific may be obtained by contacting the Licensing Department of Thermo Fisher Scientific, 5791 Van Allen Way, Carlsbad, California 92008, USA. NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by US Patent Nos. 5,677,152 and 5,773,258, and corresponding patent claims outside the US.http://www.karkarlandas.lt/fckeditor/editfiles/ads-ax2-manual.xml

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This product is for research use only. Further information on purchasing licenses from Thermo Fisher Scientific may be obtained by contacting the Licensing Department of Thermo Fisher Scientific, 5791 Van Allen Way, Carlsbad, California 92008, USA.In many cases a COA can be faxedIn many cases a COA can be faxedHot Start PCR allows fo. Keywords: Amplification, Buffers, Gas chromatography, Polymerase chain reaction, Polymerase chain reaction - quantitative Keywords: Amplification, Cloning, Gas chromatography, Gene expression, Polymerase chain reaction, Polymerase chain reaction - quantitative, Purification, Sequencing, Transcription Template purity and quality are also critical to PCR success. Sequence and p. Keywords: Amplification, Polymerase chain reaction Our PCR Selection Guide features various filters to sort by. Keywords: Genomics, Polymerase chain reaction, Polymerase chain reaction - quantitative Reproduction of any materials from the site is strictly forbidden without permission. You may now login with the following ID: Enter your Material and Lot Number in the fields above without spaces and click the search button. Select the FastStart Universal Master Mixes containing this trust-worthy polymerase. These master mixes are optimized for non-Roche qPCR-systems for both SYBR Green reactions and probe-based assays. Experience safety FastStart mixes contain dUTP for carryover prevention from previous PCR reactions, when using Uracil-DNA Glycosylase.The EagleTaq Universal Master Mix (ROX) is a ready-to-use, 2? concentrated PCR master mix for quantitative, real-time PCR hydrolysis probe reactions. It contains a special ROX reference dye which makes it suitable for all real-time instruments on which a ROX reference dye is needed for quantitative analysis.The EagleTaq Universal Master Mix (ROX) contains dUTP so that it may be used with Uracil-DNA Glycosylase (UNG) to prevent false positives arising from carryover contamination.http://autotuning-nv.ru/userfiles/ads-momentum-2009-manual.xmlThe hot-start properties allow reaction setup at ambient temperature.With this robust reagent, any PCR protocol optimization is minimized.This product is for general laboratory use. Not for use in diagnostic procedures unless otherwise indicated. This website contains information on products which is targeted to a wide range of audiences and could contain product details or information otherwise not accessible or valid in your country. Please be aware that we do not take any responsibility for accessing such information which may not comply with any valid legal process, regulation, registration or usage in the country of your origin. Your paper(s) will be evaluated for placement on lifescience.roche.com. Please separate multiple entries using comma. Your privacy is ensured. The UPL ProbeLibrary products are now available exclusively on SigmaAldrich.com. A universal primer annealing feature reduces optimization steps and allows for co-cycling of different assays. A unique combination of innovative buffer, high-performance Taq DNA polymerase, and superior hot-start technology enables exceptional PCR results, even in the toughest applications. Therefore, individual assays cannot be amplified in the same PCR run. With Platinum II Taq Hot-Start DNA Polymerase, different PCR assays can be cycled in parallel using the same protocol with universal primer annealing temperature and the extension step selected for the longest fragment to be amplified. Moreover, Platinum II Taq Hot-Start DNA Polymerase is a “fast” DNA polymerase; thus, the combination of this next-generation DNA polymerase and the universal protocol permits fast cycling of all assays in as little as 30 minutes. PCR assays using conventional PCR reagents require specific protocols for amplification of each DNA fragment because of the different primer annealing temperatures and extension steps. Therefore, with traditional PCR reagents, multiple targets often cannot be amplified together in the same PCR run.http://fscl.ru/content/deutz-allis-7085-repair-manual With Platinum II Taq Hot-Start DNA Polymerase, different PCR assays can be cycled together using one protocol with a universal primer annealing temperature and the extension step selected for the longest fragment to be amplified. Moreover, Platinum II Taq Hot-Start DNA Polymerase is a fast DNA polymerase, delivering PCR results in as little as 30 minutes. The same protocol was used for all four targets with the annealing and extension settings indicated. The size marker is Thermo Scientific ZipRuler Express DNA Ladder 2. Therefore, with Platinum II Taq Hot-Start DNA Polymerase PCR results are generally more than 2 times faster than other hot-start Taq DNA polymerases. Amplification of a 529 bp fragment from 50 ng of human genomic DNA in 50 ?L reactions for 35 cycles was carried out using Platinum II Taq Hot-Start DNA Polymerase and hot-start DNA polymerases from other suppliers: (A) Sigma-Aldrich KAPA2G Fast HotStart PCR Kit, (B) NEB OneTaq Hot Start DNA Polymerase, (C) Promega GoTaq G2 DNA Polymerase, (D) Toyobo Quick Taq HS DyeMix, (E) Roche FastStart Taq DNA Polymerase, and (F) Sigma-Aldrich JumpStart Taq DNA Polymerase. PCR product analysis in 1 TAE agarose gels is presented below the graph. The size marker is the ZipRuler Express DNA Ladder 2. Amplification of a 529 bp fragment from 0 (no template control); 0.016; 0.08; 0.4; 2; 10; 50; 250 ng of human genomic DNA were amplified in 50 ?L PCR reactions using Platinum II Taq Hot-Start DNA Polymerase or competitor DNA polymerases ( A —KAPA2G Fast HotStart, B —NEB OneTaq Hot Start, C —Promega GoTaq G2, D —Sigma JumpStart Taq, and E —Takara Taq HS Perfect Mix). The molecular weight marker is ZipRuler Express DNA Ladder 2. The molecular weight marker is ZipRuler Express DNA Ladder 2. Amplification of a 527 bp fragment from varying amounts of DNA extracted from mouse FFPE tissue samples using Platinum II Taq Hot-Start DNA polymerase. RecoverAll Total Nucleic Acid Isolation Kit for FFPE was used for DNA extraction. NTC: no template control. PC: positive control from 1 ng of purified mouse genomic DNA. The molecular weight marker is ZipRuler Express DNA Ladder 2. A separate vial of Platinum GC Enhancer is provided for specific amplification and improved yields of targets with high-GC content. The molecular weight marker is ZipRuler Express DNA Ladder 2. The enzyme’s superior performance, universal primer annealing, and fast synthesis enable generation of PCR amplicons for Sanger sequencing, with ease and simplicity. Data reported by the KB basecaller of the built-in sequencing analysis software is shown. Clear-range read length (CRL) is defined as the longest uninterrupted segment of bases at a given Quality Value (QV).Please update your browser version or choose another browser to use.Also discover different types of hot-start enzyme modifications and how to choose a hot-start DNA polymerase suitable for your PCR. Platinum II Taq Hot-Start DNA Polymerase is the next generation hot-start DNA polymerase, newly engineered for rapid, robust performance. We recommend that investigators starting new projects use Platinum II Taq DNA Polymerase so that they can benefit from its superior performance, summarized in the table below. Sci Rep 10(1):1674. Stem Cell Res 37:101451. Not for use in diagnostic procedures. This selection of papers amply illustrates how Roche Applied Science products save precious time in the development of experimental applications that range from multiplex PCR (section 8.1) to direct colony PCR (section 8.4). The authors of these papers, whether from Roche Applied Science laboratories or from outside laboratories, have one thing in common. They all found that Roche Applied Science products made their experiments easier. For example, Hermann and Foerzler (section 8.2) showed how FastStart Taq DNA Polymerase increased the sensitivity of PCR for difficult-to-amplify PCR products. Paduch (section 8.5) found that the FastStart High Fidelity PCR System made it possible to perform methylation-specific PCR, an important technique for the study of epigenetic mechanisms, without requiring extensive optimization. Ortega and Valvano (section 8.8) reported that the combination of Transcriptor Reverse Transcriptase and Taq DNA Polymerase formed an efficient two-step RT-PCR system for amplification of RNA templates with up to 70 GC content. In some cases, these products not only made their experiments easier, they made them possible. See for instance the article by Pohjanvirta (section 8.7), who found that FastStart Taq DNA Polymerase was the only hot-start enzyme that amplified all the expected products in his experimental system. 8 Applications 229 Multiplex PCR Using the FastStart High Fidelity PCR System 8.1 Multiplex PCR Using the FastStart High Fidelity PCR System Michaela Schubert1, Brigitte Hloch2 and Barbara Rueger2 Application Lab Core Biochemicals, Roche Applied Science, Penzberg, Germany 2 Global Marketing, Roche Applied Science, Penzberg, Germany 1 Introduction The Roche Applied Science FastStart High Fidelity PCR System is the product of choice for multiplex PCR. The multiplex PCR system can easily be developed and optimized in three simple, straightforward steps: X Monoplex PCR of all fragments separately X Initial multiplex PCR optimization with buffers from the PCR Optimization Kit X Final multiplex PCR optimization with the optimal PCR buffer and different additives Here, we describe a detailed, step-by-step protocol for developing such a multiplex PCR system. This protocol is suitable for many experimental systems, even when the primer set is very complex. As this article demonstrates, the system can amplify up to 18 PCR fragments simultaneously after the reaction is optimized. Materials and Methods Preparation of Template Suitable DNA could be prepared with any of a variety of Roche Applied Science reagents that are specifically designed for the preparation of high quality nucleic acids. (For details, see Chapter 3 in this manual.) RNA targets were amplified with a two-step RT-PCR procedure. For the first step, the Transcriptor First Strand cDNA Synthesis Kit and an anchored oligo(dT) primer were used to produce a cDNA template suitable for PCR. (The RT procedure was as described in section 5.4 of Chapter 5 in this manual.) An aliquot of the synthesized cDNA was used directly in each of the monoplex and multiplex reactions described below. 8 For the entire three step protocol, a total of at least one monoplex reaction for each individual primer pair and 31 multiplex optimization reactions were needed.Each of the monoplex PCRs should generate specific single products. The thermal profile below was developed for the Applied Biosystems GeneAmp PCR System 2400 thermal block cycler. Other PCR instruments may require a different profile. Increase activation time (up to 10 min) for difficult amplifications or to increase the yield of product. Increase the number of cycles to increase the yield of product. Annealing temperature depends on the melting temperature of the primers. We recommend using 1 min for 1.0 kb targets, 2 min for targets up to 1.8 kb. 231 Multiplex PCR Using the FastStart High Fidelity PCR System Step 2: Initial Optimization of Multiplex PCR with Buffers from the PCR Optimization Kit After detectable amounts of all monoplex amplification products had been generated under the same PCR conditions, these conditions had to be optimized for multiplex PCR. During the initial optimization described below, the PCR Optimization Kit (Roche Applied Science) was used to define the optimal multiplex PCR buffer. This step required a total of 17 multiplex reactions. 1 At room temperature, set up a master mix by adding the components below: The extra volume in the master mix is to compensate for pipetting losses in step 2 below. PCR Applications Manual Multiplex PCR Using the FastStart High Fidelity PCR System Step 3: Final Optimization of Multiplex PCR with the Best Performing Buffer and Different Additives After the optimal PCR buffer was identified (in the Step 2 procedure above), the procedure below was used to test the different additives from the PCR Optimization Kit. The results of this step were used to determine whether any of the additives enhanced the yield and specificity of the multiplex PCR. This step required a total of 12 multiplex reactions. 1 At room temperature, set up a master mix by adding the components below: The extra volume in the master mix is to compensate for pipetting losses in step 2 below. Results The three-step protocol above was tested on multiplex PCR systems with nine different primer sets. The results obtained with three of these primer sets is shown below. For the results obtained with all nine primer sets, see Schubert et al. (2005). The most complex primer set (an 18-plex that generated product sizes ranging from 74 to 470 bp) was tested with human genomic DNA. Figure 8.1.1 shows the results of each of the three developmental steps with that primer set. The best results were obtained with FastStart High Fidelity Buffer, extra High Fidelity Enzyme Blend and extra MgCl2 (panel C, lane 12). For panels B and C, the reagent combination(s) that gave optimal results are shown in green type. MVIII: Molecular Weight Marker VIII (RAS). The cDNA template was obtained by reverse transcription of human skeletal muscle total RNA with the Transcriptor First Strand cDNA Synthesis Kit. Only the results from the final optimization (Step 3 procedure) are shown. The reagent combination(s) that gave optimal results are shown in green type. MVIII: Molecular Weight Marker VIII. Applications 8 235 Multiplex PCR Using the FastStart High Fidelity PCR System The protocol was also tested for ability to amplify longer sequences. Discussion Any system as complex as multiplex PCR is affected by a number of factors. 8 236 Choice of PCR enzyme is obviously crucial. For multiplex PCR, the FastStart High Fidelity Enzyme System offers a number of advantages over single enzymes or other enzyme mixtures. Specifically, the optimized FastStart High Fidelity enzyme mixture transcribes more accurately (up to fourfold higher fidelity) than Taq DNA Polymerase alone and is better able to amplify sequences with high GC content (between 40 and 60, with the assistance of DMSO). The optimized enzyme mixture is also very sensitive, since we showed that it could amplify multiple targets from as little as 1 ng human genomic DNA template. In their hands, the FastStart High Fidelity Enzyme System was able to amplify all five targets from as little as 0.375 ng template. PCR Applications Manual Multiplex PCR Using the FastStart High Fidelity PCR System Successful multiplex PCR also depends greatly on the sizes of the products. It is difficult to amplify very long and short products at the same time with equivalent yields, because the polymerase will amplify the shorter products more efficiently. The purity of the primers also affects results. Only HPLC-purified primers should be used in a multiplex system. Less purified primer preparations may contain shorter oligonucleotide fragments that can contribute nonspecific bands to the final product mixture, greatly complicating interpretation of the results. Reaction additives, and particularly cosolvents were frequently found to enhance multiplex PCR results. Of the additives tried, we found DMSO and glycerol to be the most effective. Reportedly (Landre et al., 1995), DMSO reduces nonspecific priming and secondary structure (e.g., of GC-rich seqences), and facilitates strand separation by lowering the Tm of the target. Glycerol makes the Taq DNA Polymerase more resistant to heat damage, thereby increasing product yield (Aoyagi, K., 2001). Finally, successful generation of cDNA templates requires use of an anchored oligo(dT) primer, rather than a hexamer primer. Hexamer primers can interfere with the subsequent multiplex PCR. Since heterozygote samples must be unambiguously identified, excellent DNA quality is particularly crucial for applications such as mutation detection or discovery of single nucleotide polymorphisms (SNPs). Therefore, it is necessary to optimize PCR conditions for each PCR amplicon. Here, we have compared the ability of Taq DNA Polymerase and FastStart Taq DNA Polymerase to amplify difficult (GC-rich) DNA templates in the presence and absence of cosolvents, starting with different amounts of DNA. Reactions were performed in a thermal cycler (MJ Research Tetrad, Watertown, MA). Results and Discussion In reactions with either Taq DNA Polymerase or FastStart Taq DNA Polymerase, it was not possible to obtain the desired 540-bp amplicon in sufficient amounts without adding DMSO or GC-rich solution. Figure 8.2.1. Amplification of a 540-bp fragment of the hydroxytryptamine receptor 2C (with a GC content of 65) using FastStart Taq DNA Polymerase and Taq DNA Polymerase. Different amounts of DMSO or GC-rich solution were added. High concentrations (more than 2) of DMSO inhibit the activity of Taq DNA Polymerase, but do not inhibit the activity of FastStart Taq DNA Polymerase. FastStart Taq DNA Polymerase, when used with the specially developed GC-rich solution, seems to be the most suitable enzyme for templates with a high (65) GC content. Applications 8 239 Specific Amplification of Difficult PCR Products from Small Amounts of DNA. Figure 8.2.2. Dilutions of human genomic DNA were amplified in the presence of 2 DMSO or 2 GC-rich solution. FastStart Taq DNA Polymerase showed greater sensitivity (i.e., a visible PCR product was obtained from only 100 pg of starting DNA). Arrows mark the threshold of detection for each enzyme-cosolvent combination. 8 240 To determine the sensitivity of both enzymes, dilutions of human genomic DNA (50 pg, 100 pg, 200 pg, 500 pg, 1 ng, 2 ng, 5 ng, 10 ng, 20 ng) were amplified in the presence of 2 DMSO or 2 GC-rich solution (Figure 8.2.2). The threshold of detection is defined as the amount of starting DNA that generates a visible PCR signal (i.e., presence of the 540-bp fragment on the gel; see arrows). Taq DNA Polymerase produced a visible PCR product in 2 DMSO from as little as 10 ng template DNA, and in 2 GC-rich solution from as little as 2 ng DNA. FastStart Taq DNA Polymerase showed at least tenfold greater sensitivity, generating visible PCR products from only 100 pg of DNA template. These results are in agreement with the results obtained with different concentrations of cosolvents. The higher sensitivity of FastStart Taq DNA Polymerase can be explained by its robustness in the presence of additives (e.g., DMSO or GC-rich solution). FastStart Taq DNA Polymerase did not seem to be inhibited by these cosolvents, nor did the solvents slow the extension rate of the enzyme. PCR Applications Manual Specific Amplification of Difficult PCR Products from Small Amounts of DNA. Summary We have successfully used FastStart Taq DNA Polymerase to obtain clean, specific PCR products, suitable for sequencing, from small amounts (as little as 100 pg) of GC-rich template DNA. In addition to the advantage of higher specificity offered by a hot start enzyme, FastStart Taq Polymerase has an additional advantage; it is not inhibited even by high concentrations of cosolvents (DMSO and GC-rich solution). Therefore it is superior to Taq DNA Polymerase for this application. Products from Roche Applied Science used to generate these results: Taq DNA Polymerase; FastStart Taq DNA Polymerase. However, if only small quantities of starting material are available, it is important to amplify the genes with great specificity and sensitivity. We have therefore undertaken a comparative study of commercially available reverse transcriptases and hot start Taq DNA Polymerases to identify the optimal enzyme combination for quantification of gene expression in a small quantity of cells isolated by LCM. Total RNA was extracted from each sample with a commercially available kit, then divided into three equal parts. Each reaction was performed according to the manufacturer’s instructions. The reaction with FastStart Taq DNA Polymerase contained 0.8 unit of enzyme and a final MgCl2 concentration of 2 mM. The resulting 300-bp PCR products were electrophoresed in parallel through a 2 agarose gel in the presence of a SYBR green stain, then visualised under UV light. PCR Applications Manual FastStart Taq DNA Polymerase Is Ideally Suited for RT-PCR of Laser Captured Microdissected Material Results and Discussion To compare the efficiency of several reverse transcriptases and hot start Taq DNA Polymerases in two-step RT-PCRs, total RNA was extracted from luminal epithelial LCM samples of a mouse uterus. Three different reverse transcriptases were used for cDNA synthesis. Five different hot start Taq DNA Polymerases, including FastStart Taq DNA Polymerase, were used for subsequent PCR amplification of GAPDH. PCR amplifications were performed in parallel. GAPDH cDNA was amplified in each case but the different combinations of reverse transcriptases and hot start Taq DNA Polymerases produced varying degrees of efficiency and specificity (Figure 8.3.1). The amount of GAPDH amplification with FastStart Taq DNA Polymerase was generally as good as or better than amplification with hot start Taq DNA Polymerases from other suppliers. However, non-specific amplification was detected in the majority of PCRs that used hot start Taq DNA Polymerase from other suppliers, whereas it was minimal in PCRs that used FastStart Taq DNA Polymerase. FastStart Taq DNA Polymerase was therefore chosen for experiments involving PCR amplification of RNA extracted from LCM material. Figure 8.3.1. Comparison of reverse transcriptase and hot start Taq DNA Polymerase efficiency in two-step RT-PCRs.The template RNA was obtained from mouse uterine luminal epithelium cells isolated by laser capture microdissection. The 300-bp PCR product, a GAPDH amplicon, was obtained after 40 PCR cycles. FastStart Taq DNA Polymerase has also successfully amplified targets present at a lower abundance than GAPDH in LCM material, such as estrogen receptors alpha and beta (results not shown). Summary 8 We have successfully used FastStart Taq DNA Polymerase to amplify gene expression targets from limited amounts of cells that were isolated by laser capture microdissection. This product provides superior sensitivity and less nonspecific amplification than other hot start Taq DNA Polymerases. Product from Roche Applied Science used to generate these results: FastStart Taq DNA Polymerase. For information on ordering this product, see the Appendix. Direct colony PCR offers a fast screening method that circumvents the need for template purification before PCR and allows the rapid characterization of multiple clones. For this application a robust but specific and sensitive PCR system is needed. The Roche Applied Science FastStart PCR Master (a ready-to-use, 2? concentrated reaction mix for hot-start PCR) provides such a system for direct colony PCR. In this paper, we describe the use of the FastStart PCR Master for the amplification and cloning of mRNA. An overview of the workflow for the technique, which includes direct colony screening, is presented in Figure 8.4.1. 8 244 PCR Applications Manual Cloning of mRNAs and Rapid Screening by Direct Colony PCR with the FastStart PCR Master K562 cells HIGH PURE RNA Isolation Kit RNA Transcriptor First Strand cDNA Synthesis Kit cDNA FastStart PCR Master or Pwo SuperYield Polymerase DNA HIGH PURE PCR Product Purification Kit Purification of PCR product T4-DNA Polymerase Polishing HIGH PURE PCR Product Purification Kit Purification of PCR product PCR Cloning Kit (blunt-end) Procedure for vector linearization and ligation Procedure for transformation Ampicillin Counting of colonies FastStart PCR Master or Pwo SuperYield Polymerase Colony PCR Figure 8.4.1. Workflow strategy. Materials and Methods All reagents (except for growth media and competent cells), enzymes and kits were supplied by Roche Applied Science. Purification of Total RNA 8 Total RNA was obtained from K562 cells with the HIGH PURE RNA Isolation Kit as described in the package insert. The desired target of the amplification was a 380-bp fragment of the human interleukin gene (IL-2). Purification and Cloning of PCR Product The PCR product from each reaction was purified with the HIGH PURE PCR Product Purification Kit as described in the package insert. The purification procedure was functionally very similar to the RNA purification described above, since it also involved a HIGH PURE Filter Tube, but required a DNA-specific Wash Buffer and different centrifugation times. 8 246 Subsequently, the PCR product generated with the FastStart PCR Master was polished with T4 DNA Polymerase to generate blunt-ended PCR fragments. Note: Since Pwo SuperYield DNA Polymerase generates blunt-ended products, the PCR product generated with Pwo SuperYield DNA Polymerase did not require polishing. The purified amplification product from each PCR was cloned into a pCAPs cloning vector with the PCR Cloning Kit (blunt-end). Vector restriction and ligation were performed according to the package insert of the kit. Direct Colony PCR Ten clones derived from each cloned PCR product were randomly picked with sterile toothpicks. The 380-bp fragment of the IL-2 gene was amplified using either FastStart Master Mix (FS) or Pwo SuperYield DNA Polymerase (PWO) according to the package insert of each polymerase. The amplification efficiency of the FastStart Master Mix was comparable to that of the Pwo SuperYield DNA Polymerase. M, Molecular Weight Marker VIII. Agarose gel analysis showed that the polymerases consistently amplified a 380-bp fragment (Figure 2). The FastStart PCR Master and the Pwo SuperYield DNA Polymerase yielded comparable amounts of amplification product. PCR for Colony Screening More than 50 colonies were obtained from each cloning experiment. Comparable numbers of colonies were obtained from cloned PCR products that were generated with the two DNA polymerases. 8 248 Figure 8.4.3. Colony PCR of clones that contained plasmid and inserts generated with the FastStart PCR Master. Direct colony PCR with the FastStart PCR Master was used to amplify the cloned 380-bp fragment from the IL-2 gene. The gel shows that all randomly selected colonies carried the ligation product. Figure 8.4.4. Colony PCR of clones that contained plasmid and inserts generated with Pwo SuperYield DNA Polymerase. The gel shows that all randomly selected colonies carried the ligation product. PCR Applications Manual Cloning of mRNAs and Rapid Screening by Direct Colony PCR with the FastStart PCR Master The FastStart PCR Master was used for direct colony PCR. All 20 randomly selected clones contained the 380-bp insert ligated into the plasmid vector pCAPs (Figures 3, 4). Consequently, the cloning experiment succeeded regardless of the polymerase used to amplify the original cDNA and produce the 380-bp insert. Pwo SuperYield DNA Polymerase is a proofreading polymerase while FastStart Taq DNA is not.