【原创】lentivirus calcium phosphate transfection protocl
丁香园论坛
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calcium phosphate transfection protocl：
1. Split 293FT cells about 30% confluence, overnight.
2. Thaw undefinedHBS room temperature (not in 37 C).
3. For 10cm dish:
1.5ml tube, 4ug packaging plasmid A, 4ug packaging plasmid B, 4ug expression plasmid, 61ul of 2M CaCl2,
Mix properly, and then add distilled water to total volume of 0.5ml
4. Add 500ml undefinedHBS to the tube, mix gently. Wait for 15min ~30min until small white precipitation appear.
5. Add the mixture to cells slowly. Do not mix the medium, but simply carry the dish to the incubator (this mixes it ),place at 37% CO2 overnight
6. After o/n, change the medium, continue to culture for 48hours.collect the medium, from which you can spin down and purify the virus.
undefinedHBS:
8g NaCl
0.2g Na2HPO4-7 H2O (0.14 if using dehydrate,[phosphate] must be 1.5mM)
6.5g HEPES
Add distilled water to the total volume of 500ml,
Adjust PH to 7.12
Store at -80 C
注意事项：
1. 293FT 应该是 invitrogen提供的，所以他们建议使用，个人比较过，确实比293T好点，但差别不是非常大。
2. HBS 的ph值非常重要，需要严格控制，解冻后的HBS 不可再用。
3.一般package plasmid 有一个是vsvg，可能有益于慢病毒的稳定性，目前还在测试中。
4.一般sh等小序列直接用medium都可以，效力也比较高，超过2Kbp的最好纯化一下，至少高速离心一下。
5.试过用DNA-LIPOFECTAMINIE 2000 转染，效果确实比calcium好很多，当然，价格也高了很多，经费足的，可以选用。
1. Split 293FT cells about 30% confluence, overnight.
2. Thaw undefinedHBS room temperature (not in 37 C).
3. For 10cm dish:
1.5ml tube, 4ug packaging plasmid A, 4ug packaging plasmid B, 4ug expression plasmid, 61ul of 2M CaCl2,
Mix properly, and then add distilled water to total volume of 0.5ml
4. Add 500ml undefinedHBS to the tube, mix gently. Wait for 15min ~30min until small white precipitation appear.
5. Add the mixture to cells slowly. Do not mix the medium, but simply carry the dish to the incubator (this mixes it ),place at 37% CO2 overnight
6. After o/n, change the medium, continue to culture for 48hours.collect the medium, from which you can spin down and purify the virus.
undefinedHBS:
8g NaCl
0.2g Na2HPO4-7 H2O (0.14 if using dehydrate,[phosphate] must be 1.5mM)
6.5g HEPES
Add distilled water to the total volume of 500ml,
Adjust PH to 7.12
Store at -80 C
注意事项：
1. 293FT 应该是 invitrogen提供的，所以他们建议使用，个人比较过，确实比293T好点，但差别不是非常大。
2. HBS 的ph值非常重要，需要严格控制，解冻后的HBS 不可再用。
3.一般package plasmid 有一个是vsvg，可能有益于慢病毒的稳定性，目前还在测试中。
4.一般sh等小序列直接用medium都可以，效力也比较高，超过2Kbp的最好纯化一下，至少高速离心一下。
5.试过用DNA-LIPOFECTAMINIE 2000 转染，效果确实比calcium好很多，当然，价格也高了很多，经费足的，可以选用。
