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Amplification of Epstein-Barr Virus Exon C with Difficult Primers ,,, Using Eppendorf Mastercycler gradient

g reactions. For this reason, primers flanking exactly the 5'-terminal and the 3'-terminal of each exon were synthesized in order that the cloned amplimer will be in-framed during subsequent clonings (Fig.1). In so doing, we were restricted to only one choice for each primer. Exons A and B were successfully amplified by their respective primers at the calculated annealing temperature using a conventional thermal cycler. However, this was not the case for Exon C. The properties of primers used for amplifying Exon C are tabulated in Table 1.



Table 1: Properties of primer CF and CR specific for Exon C of LMP-1 Methods

During optimization for primers CF and CR, we applied the general rule of starting with annealing temperatures around 5C below the calculated melting temperature (Tm ) and titrating for optimal MgCl2 concentrations. The Tm of both primers were recommended by the manufacturer (Operon, Inc., Alameda, CA, USA) calculated using the standard formula. Using a conventional thermal cycler, we were unable to amplify Exon C despite attempts over a range of annealing temperatures (52C to 65C) and MgCl2 concentrations (1 mM to 4 mM). This problem was solved when we used the temperature gradient function of the Eppendorf Mastercycler gradient.4,5

By performing a wild search for the correct annealing temperature, we subjected our amplification reaction using primers CF and CR (Table 1) over a temperature gradient of 20C (48C to 68C) at a constant MgCl2 concentration of 2 mM. Template EBV DNA was extracted from B95.8 marmoset lymphocytes cell line. PCR was carried out in 50 l reactions with the f
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