Establishment and optimization of SCoT-PCR reaction system for Lucuma nervosa A.DC
To establish and optimize the SCoT-PCR reaction system for Lucuma nervosa A.DC,factors affect-ing SCoT-PCR reaction such as template DNA,Mg2+,dNTPs,primers and Taq DNA polymerases were opti-mized using L.nervosa A.DC(Xiantao No.1)as research material and SCoT1 as primer by orthogonal exper-iment of L16(45).DNA samples from three germplasms of L.nervosa A.DC located far away from each other were selected as templates to verify the optimized reaction system.The results showed that the effects of vari-ous factors on the SCoT-PCR reaction system of L.nervosa A.DC were different,but not significant.The most important factor was the concentration of Taq DNA polymerase,followed by the concentrations of dNTPs,template DNA,primers and Mg2+.The optimal SCoT-PCR reaction system(20 μL)of L.nervosa A.DC was 80 ng template DNA,3 mmol/L Mg2+,0.25 mmol/L dNTPs,0.4 μmol/L primer,2 U Taq DNA polymerases.With this system,the clear,bright,abundant and stable bands could be amplified from three germplasms,which indicated that the optimized SCoT-PCR reaction system was suitable for use with L.nervosa A.DC SCoT molecular markers.
Lucuma nervosa A.DCSCoT-PCRorthogonal experimentTaq DNA polymerasedNTPs con-centration
万方数据
维普
