Reagents for PCR

The reagents required for PCR, apart from the DNA template itself, are the DNA polymerase enzyme, an appropriate pair of primers and the four nucleotides (dNTPs). For the PCR to be successful, the reaction conditions have to be carefully controlled, including pH, ionic strength and additives. The reagents used for PCR are outlined in more detail below. 

Primers are short oligonucleotides, which are complementary to the ends of the target sequence. Two distinctive primers are used for a PCR amplification a forward primer and a reverse primer (Fig. 6.2). Each hybridises to one of the two strands of the original dsDNA molecule (see Fig. 6.4, cycle one). DNA synthesis always proceeds in the direction from 5 to 3. The first nucleotide to be incorporated reacts with the free 3 -hydroxyl group of the primer. The 5 -end of the primer is blocked. 

The set of primers has to be designed specifically for the sample DNA. The primer length is usually 10-30 base pairs (bp) and their complementary sequence must be unique in the template. Additionally, there should be no intra or inter primer complementarity in order to avoid the formation of primer-dimers. Ideally, the number of each base in the primer is relatively equal. Unusual sequences such as long stretches of polypurines or polypyrimidines and repetitive sequences must be avoided. The melting temperature for both primers should be similar and lie between 55 and 80 °C. 

The four deoxynucleotide triphosphates, dATP, dCTP, dGTP and dTTP, are the building blocks for DNA synthesis (see section 1.2.1.1). The reaction mixture must contain an excess of these dNTPs as they deplete during PCR. The concentration of each dNTP should be equal. 

The buffer pH and ionic strength is chosen according to the polymerase used. The ionic strength has a crucial influence on the specificity of the PCR. A typical buffer system has an ionic strength of about 50 mM and consists of Tris-HCl, pH 8.3, with KCl or NaCl. 

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DNA was isolated from the gel block using the DNA extraction kit (Cytokine, Russia). DNA added to the tube containing the reagents for PCR (DNA-Technology, Russia) and amplified in 35 cycles (30s 93°C, 30s 59°C and 30s at 72°C). For subsequent analysis 5 pi of the product was analyzed by gel electrophoresis as described above. 

The quality and consistency of reagents for PCR is constant between lots. However, antibodies produced by different individuals or species of animals differ in... 

Check the stock of reagents for reverse transcriptase and PCR reaction, RNase-free DNase, Kim wipes, parafilms, plastic coverslips. 

Before starting experiments with human or animal tissue samples, it is extremely important to optimize in vitro experimental conditions. With a purified template nucleic acid, standardize RT and PCR conditions. Check the specificity and crossreactivity of primers and probes. Sometimes it is necessary to alter MgCl2 concentration under in situ reaction conditions. The blocking reagent for filter hybridization could be different than the in situ protocol. (I use 1 % purified casein solution for filter hybridization and 3% BSA for in situ signal detection.)... 

Preparation of the Premix solution for PCR amplification add to an Eppendorf tube the following reagents in this order ... 

The major drawback of the real-time method IPCR is the need for a specialized real-time cycler, preferably compatible to the microplate format. With the increasing distribution of these machines, there is also an emerging range of new opportunities for real-time IPCR. The need of an additional fluorescent probe during PCR is compensated for by the fact that all materials and reagents for post-PCR processing were no longer required. However, the need for separate discriminable fluorescence probes reduces the usefulness of real-time detection in multiplex IPCR applications. 

A similar PDMS valve control layer was used to achieve rotary liquid pumping for PCR [357]. In another report, a similar valving method was used to deliver cells and to introduce reagents for cell reactions. Solutions were pumped at 5-60 Hz to achieve a linear flow rate of 300-1000 pm/s [368]. 

Fig. 2. Effect of DNA concentration and number of PCR cycles on RAPD analysis, shown on ethidium bromide-stained agarose gels, using template DNA from a single individual titmouse (Parus bicolor) and the 10-base primer AP5a4 (5 CTGTTGCTAC 3 ). (A) Various concentrations of template DNA amplified through 45 cycles of PCR. Lanes 1-11 contain 100, 50, 20, 10, 5, 2, 1, 0.1, 0.05, 0.01, and 0.005 ng of template DNA, respectively, in 25-/

The second most common source of contamination is contemporary DNA present in chemicals used for DNA extraction or for the PCR. All reagents should be stored in small aliquots to facilitate replacing them when contamination occurs. Solutions and chemicals should be screened by performing control extracts and PCR amplifications as discussed above. Sometimes extensive testing is required before, for example, a source of uncontaminated water can be found. Once a clean source is identified, it should be aliquotted to avoid future contamination of the entire batch and used exclusively for PCR of ancient DNA. 

Figure 2. Coronin 1C in melanoma. A) Immunohistochemical staining of the indicated tissue samples for Coronin 1C. S m sections from melanoma tumors were stained with a new Coronin 1C specific mAb (Roadcap and Bear, unpublished reagent) for 30min at 37°C with a steam heat-induced epitope retrieval, using the streptavidin/biotin method with an alkaline phosphatase label and Permanent Red (Dako) as the chromogen and then stained with hematoxylin. B) qRT-PCR analysis of the indicated melanoma cell lines treated with the Erk inhibitor U0126 or vehicle control for 24 hours.Dotted line indicates lx threshold.

All reagents used for the PERT assay must be free of any contaminating MS 2-DNA. To avoid carryover contamination, set up at least two different rooms for work with materials before and after PCR. Use different sets of equipment, chemicals, and disposables. If possible, avoid the use of pH probes and spatula. If glass ware is used, do not have them washed in a central facility (where it can get contaminated), wash and bake it personally (at 240°C for 5 h). It may be a good idea to have a friend at a different location (where no work with MS2 is carried out) who can prepare solutions. Use only positive displacement pipetes or pipet tips with filters especially for PCR. The published recommendations in ref. 17 are useful. 

Thermal cycling polymerase chain reaction (PCR) machine and reagents for performing PCR GeneAmp PCR Reagent Kit (Perkin-Elmer, Foster City, CA). 

Reagents and equipment for PCR, restriction digests, and DNA Hgations. Reagents to vahdate expression of fusion proteins, such as antibodies for flow cytometry and/or Western blotting. 

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