百合鳞片DNA提取及RGA-PCR体系的优化

DNA Extraction and Optimization of RGA-PCR Reaction System for Bulbs of Lilium

以百合鳞片为材料,采用改良的CTAB法获得了高质量的基因组DNA。通过影响PCR反应各因子的优化,建立了适合百合的RGA-PCR反应体系:25μl体系中含30 ng模板DNA、2.5 mmol/L MgCl2、0.2 mmol/L dNTPs、0.6μmol/L引物及1.5 UTaq酶。扩增程序为:94℃预变性5 min,94℃变性1 min,44℃退火1 min,72℃延伸2 min,40个循环;最后72℃延伸7 min。利用该体系对35个百合品种进行RGA-PCR,表明该反应体系具有较好的稳定性和可靠性。

High quality genomic DNA was obtained from lily bulbs using improved CTAB method.Different factors that affected amplification reaction were optimized to establish the RGA-PCR reaction system and procedure of lily.The reaction were performed in a volume of 25 μl containing 30 ng of DNA,2.5 mmol/L MgCl2,0.2 mmol/L dNTPs,0.6 μmol/L primer and 1.5 U Taq DNA polymerase.The reaction procedure was as follows： pre-denaturing at 94 ℃ for 5 min,40 cycles of denaturing at 94 ℃ for 1 min,annealing at 44 ℃ for 1 min and extending at 72 ℃ for 2 min,extending at 72 ℃ for 7 min after the cycles.The optimized RGA-PCR system was tested on 30 lily cultivars and shown to be steady and reliable.