Editing PCR Master Mix

10×PCR reaction buffer consisted of MgSO 4 20Mm, KCl 100 mM, (NH 4 ) 2 SO 4 80 mM, Tris-HCl (pH = 9.0) 100 mM and NP-40 0.5% (v/v). 5×compatible dye mix included 100g Ficoll400/L, 0.1 g cresol red/L, 0.8 g tartrazine/L, Gelred 1.25× (Original concentration is 10000× in DMSO; Biocompare, USA). 2×One-stop PCR master mix (2×Red mix) was prepared as follows: 10×PCR reaction buffer, 2 μl; 40 mM dNTP (10mM each), 0.5 μl; Taq DNA polymerase, 0.2 μl (1 unit); 5×compatible dye mix, 4 μl; PCR-grade water, 3.3 μl. The final pH of 2×Red mix was 7.5, when all the compounds were completely<br/>dissolved at 25°C. A typical 20 μl one-stop PCR mixture was composed of 10 μl 2×One-stop PCR master mix, 0.5 μl forward and reverse primer mix (10 μM total), 0.5 μl cDNA, 9 μl PCR-grade water. A 20 μl traditional PCR mixture included 10×PCR reaction buffer, 2 μl, 40 mM dNTP (10 mM each), 0.5 μl; Taq DNA<br/>polymerase, 0.2 μl (1 unit), 0.5 μl forward and reverse primer mix (10 μM total), 0.5 μl cDNA, 16.3 μl PCR-grade water. Three PCR kits (Taq DNA polymerase, DreamTaq DNA polymerase, 2× Blue master mix) were purchased from Shenergy (Shanghai, China), Fermentas (Lithuania) and DBI (Shanghai, China), respectively. All cycling condition were standardized as: 94°C, 2 min; 30 cycles of (94°C, 15 s, 55°C, 15 s, 72°C, 1 min); 72°C, 5 min. All cyclings were <br/>carried out in a Minicycler machine (MJ Research, USA). Unless indicated elsewhere, the products of traditional PCR and DNA ladders were separately mixed with 1×loading buffer containing 5% glycerol, 0.04% brom phenolblue, 0.04% xylene cyanol, 0.25× Gelred, before loading for electrophoresis.




[[Category:Protocols]]