BamHⅠ+XhoⅠ双酶切质粒电泳弥散原因初探

Preliminary exploration on the causes of plasmid smear produced by double digestion with Bam HⅠ and Xho Ⅰ

目的探讨TaKaRa公司(中国大连)快速限制酶Bam HⅠ+XhoⅠ37℃双酶切质粒p ET-28a、p ET-28a-SDHA产生弥散的原因。方法比较4种条件下酶切对弥散的影响:(1)Bam HⅠ+XhoⅠ37℃双酶切Elution Buffer(EB)洗脱或水洗脱的质粒;(2)NdeⅠ+XhoⅠ37℃双酶切EB洗脱或水洗脱的质粒;(3)先Bam HⅠ30℃酶切,再XhoⅠ37℃酶切;(4)Bam HⅠ分别于30℃、37℃单酶切。结果 (1)Bam HⅠ+XhoⅠ37℃双酶切EB或水洗脱的质粒均产生严重弥散;(2)NdeⅠ+XhoⅠ37℃双酶切EB洗脱或水洗脱的质粒无弥散;(3)Bam HⅠ30℃酶切+XhoⅠ37℃酶切,无弥散;(4)Bam HⅠ30℃单酶切无弥散,37℃单酶切严重弥散。结论 Bam HⅠ+XhoⅠ37℃酶切质粒电泳弥散是Bam HⅠ在此温度下的星活性所致。

Objective To explore the causes of the smear of plasmids（ p ET-28 a and p ET-28 a-SDHA）,doubly digested by Bam HⅠand Xho Ⅰ（ Takara,Dalian,China） at 37 ℃. Methods The effects of plasmid digestion under four conditions on smear were compared. First,Bam HⅠ and Xho Ⅰ codigested plasmids eluted by elution buffer（ EB） or water at 37 ℃. Second,Nde Ⅰ and Xho Ⅰcodigested plasmids at 37 ℃. Third,plasmids were digested by Bam HⅠ at 30 ℃ and followed by Xho Ⅰ at 37 ℃. Fourth,plasmids were digested by Bam HⅠ at 30 ℃ or 37 ℃. Results The serious smear appeared in plasmids after eluted by EB or water,and digested by Bam HⅠ and Xho Ⅰ at 37 ℃. But the smear did not appear in the above-mentioned plamids digested by Nde Ⅰ and Xho Ⅰat 37 ℃,or by Bam HⅠ at 30 ℃ plus Xho Ⅰ at 37 ℃. What＇s more,the smear did not appear in plasmids digested by Bam HⅠ at30 ℃,but did when digested at 37 ℃. Conclusion The smear of plasmids doubly digested by Bam HⅠ and Xho Ⅰ at 37 ℃ is caused by the star activity of Bam HⅠ digestion at 37 ℃.