PDF《Technical Manual》

96 well

24 well

12 well

6 well

36 mm

60 mm

100 mm DNA-HilyMax 复合体调制条件 细胞培养条件0.3 cm2 1.9 cm2 3.8 cm2 9.2 cm2 8.0 cm2 21.0 cm2 58.0 cm2 0.1 ml 0.5 ml 1.0 ml 2.0 ml 2.0 ml 5.0 ml 15.0 ml

10 ?l

30 ?l

60 ?l

120 ?l

120 ?l

300 ?l

900 ?l 0.1-0.3 ?g 0.5-1.5 ?g 1.0-3.0 ?g 2.0-6.0 ?g 2.0-6.0 ?g 5.0-15.0 ?g 15.0-45.0 ?g 1:2-1:7 1:2-1:7 1:2-1:7 1:2-1:7 1:2-1:7 1:2-1:7 1:2-1:7 表2. 不同培养板的转染条件病毒包装转染 (以HEK293T细胞为例): 1. 配制DMEM+10% FBS

100 ml,置于37℃预热. 2. 转染前24 h传代HEK293T细胞 a) ,当细胞40-60%融合时,开始转染. 3. 配制OptiMEM并加入500 ?l至离心管. 4. 将病毒质粒加入到以上离心管中混匀,各质粒与培养基添加量请参考表3. 5. 按照质粒 (?g) : HilyMax (?l)为1:2.5 -1:3.5 b) 的比例加入HilyMax工作液至离心管并轻柔混匀, 实际添加量请参考表4. 6. 混合液静置15 min c) ,加入500 ?l的完全培养基(DMEM+10% FBS) 混匀后,加至HEK293T细胞的 培养皿中,"+"字形上下左右混匀培养基5次. 7. 37℃,5% CO2条件下培养 d) ,24 h后每皿补加新鲜培养基10 ml,继续培养60-72 h后,收集含病毒 的培养基. a) 工具细胞可根据实验需求进行替换. b) 如果使用其他培养板,质粒和HilyMax比例不变,添加量需根据表1换算所得. c) 混合液静置过程中,同时从培养箱中取出需转染HEK293T细胞培养皿,换新鲜、预热的完全培养基. (换液时加9 ml新鲜培养基,用含血清培养基洗细胞) d) 若要进一步减轻转染试剂的毒性,建议在培养了4-8 h内换新鲜培养基10 ml. * 以上实验步骤与数据仅供参考,具体条件可根据实验需要自行调整. 表4. HilyMax 工作液添加量HilyMax 25-35 ?l Plasmids Weight of Plasmids Volume of OptiMEM pMD.G

2 ?g Δ8.91

3 ?g

500 ?l Target Gene Vector/Ctrl Vector

5 ?g 表3. 质粒与培养基添加量(10 cm 皿) FAQ 参考文献 如果您需要更多的信息或者有任何问题可以通过以下的方式联系我们: 上海 上海市零陵路899号飞洲国际广场27楼J座 邮编:200030

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