RT-PCR Analysis
Solutions
10X RT Buffer 10X PCR Buffer
100 mM Tris pH 9.0
500 mM KCl
1% Triton X-100
25 mM MgCl2 use at a concentration of 1.5 mM Lysis Solution 4M GuSCN 250 g guanidine thiocyanate 25mM Na citrate 7.0 17.6 ml 0.75M Na citrate pH 7.0 0.5% Sarkosyl 26.4 ml 10% Sarkosyl add 293 ml Q before use, add 72ml bMEbME added just prior to use. If collecting several samples hold tubes on ice. These can be stored for years at -80°C.ml Lysis Solution and add 300 ml isopropanol. Hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.ml DEPC treated Q and store at -80°C.a-32P dATP Water Saturate Phenol thaw 500 ml phenol add 0.5 g hydroxyquinolin add 500 ml Q mix and allow phases to separate at room temperature repeat 2 times and store at 4°C 3M NaOAc 5.2 24.6 g NaOAc (anhydrous) pH to 5.2 with acetic acid up to 100 ml Q rna isolation • Add tissue to 400 microliters lysis solution with • Add 1 microliter glycogen, 30 microliters 3M NaOAc 5.2 and 500 microliters water saturated phenol. Mix by gentle inversion and add 100 microliters chloroform. • Mix by inversion and hold on ice for 15 minutes. • Spin for 10 minutes at 4°C and remove the aqueous phase. Add 500 microliters isopropanol, hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry. • Resuspend the pellet in 300 • Resuspend the pellet in 10 reverse transcription reaction • Add 5 microliters RNA to a sterile siliconized eppendorf tube along with 1 microliter of oligo dT (0.25 mg/ml). • Heat to 65° for four minutes and immediately transfer to ice. • Quickly spin the RNA/oligo dT and add 14 microliters of the RT cocktail: 2 microliters 10X RT buffer 1 microliter 1 mg/ml BSA 1 microliter 20 mM DTT 0.5 microliters RNaseIN 0.4 microliters 25 mM dNTPs 0.2 microliters AMV RT 9 microliters Q • Incubate at 48° for 30 minutes, dilute 5 fold and store at -20°. pcr reaction • Dilute cDNA (5 fold) and use between 1-5 microliters of cDNA per PCR reaction. • Add the following PCR cocktail per tube: 2.5 microliters 10X PCR buffer 1.5 microliters 25 mM MgCl2 1 microliter each primer (10pmol/microliter; 55° Tm) 0.2 microliters 25 mM dNTPs 0.025 microliters 0.02 microliters Taq polymerase (protocol T.2) 14 microliters Q • Perform PCR using the following cycle parameters: 94° for 4 minutes, (94° for 30 seconds, 55° for 30 seconds, 72° for 30 seconds) x n, 72° for 5 minutes. • The number of cycle should be determined empirically to find the linear range of amplification. Procedure
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