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Condition of amplification:I Cycle Denaturation 950C for 2 minutes20 CyclesDenaturationHybridization950C for I minute550C for I minutePolymerisationnoc for I minuteI CycleElongationnoc for 5 minutes4QC for overnight3.6.2.4.3 Second PCR: Single step: Each product of the first peR wastested against the four type-specific primers as follows:Tube I contained a mixture of 2lJI of the first PCR product (product A) diluted1:100 in PCR grade water. 31-1! of DSI, 3 fll of TSI, 51-11 reaction buffer (lOx), 4[1! ofdNTP, 3[1l ofMgCl2, 0,81-11 of Tag polymerase and 30,2[11 of PCR water. Tube 2contained 2[11 of product A, diluted I: 100 in PCR grade water, 31-11 of DS 1, 3ftl ofTS2, 5ft\ of reaction buffer (lOx), 4ft\ of dNTP, 3[l1 of MgC!2, 0.8[l1 of Taqpolymerase and 30.21-11 of PCR water. Tube 3 contained 2lJ! of product A diluted1;100 in PCR grade water, 31-11 ofDSI, 3ftl ofTS3, 5[11 of reaction buffer (IOx), 4JlIof dNTP, 3ftl of MgCI2, 0.8ftl of Taq polymerase and 30.2[l1 of peR water. Tube 4contained 2ft! of product A diluted 1:100 in PCR grade wter.Bul ofDSI, 3ftl of TS4,Sill of reaction buffer (IOx), 4ftl of dNTP, 3ft! of MgC!2, 0.8ft! of Taq polymeraseand 30.2[11 of PCR water. Negative control tube contained 2[1! of PCR water, 3[11 ofDSI, 3[11 of TSI, 5[11 of reaction buffer (lOx), 4ft! of dNTP, 3[1l of MgCI2, 0.8[11 ofTaq polymerase and 30.2ftl of PCR water. Positivc control tube contained2ft! offirst PCR product for DEN-I diluted 1:100 in PCR grade wter.Sul of DSI, 3ftl ofTSI, 5 [11 of reaction buffer (1Ox), 4ftl of dNTP, 3[11 of MgCI2, 0.8JlI of Taqpolymerase and 30.21l1 of PCR water.79