北川白山羊血液中总DNA的不同提取条件对PCR的影响

对35只北川白山羊血液中总DNA提取并根据普通牛已知的κ-CN基因序列设计一对引物进行扩增,对影响PCR反应结果的因素进行了比较分析。结果表明:蛋白酶K终浓度为0.05 mg/mL,饱和酚—氯仿抽提3次、每次30 min,采用乙醇沉淀DNA时,DNA产量最低,PCR反应无特异条带;蛋白酶K终浓度为0.1 mg/mL,饱和酚-氯仿抽提1次、每次5 min,分别采用超速离心和乙醇沉淀DNA时,DNA产量最高,PCR反应有特异条带(350 bp),但前者出现引物二聚体,故作PCR反应时选择后者沉淀最佳。

Effect of the Different Conchtions of Genome DNA Extraction on PCR in Blood of Beichuan White Goat(Oreamnos americanus)

Genome DNA was extracted from 35 blood samples of Beichuan White Goat(Oreamnos americanus),and according to kappa-casein gene sequence of bovine(Bos),a pair of primer was designed for PCR amplification.The effecting factors on PCR were analyzed.The results show that when the final concentration of protease K is 0.05 mg/mL,phenol-chloroform extracts three times by thirty minutes at the same time,and deposits DNA with ethanol,DNA production is less than other group.There is no special band in PCR.And when the final concentration of protease K is 0.1 mg/mL,phenol-chloroform extracts DNA once by five minutes at a time,and deposits DNA adopting ethanol,DNA production is the highest.There appears special band in PCR.But the former has primer dimer.So the best selection is the latter to PCR.