一头撞死……我的PCR经历……
superviser 临走的时候丢给我们17对primer, 让我们测试他们是否可行。
Group member情况：
我，正在上LSM2202,学了PCR的一点皮毛。
QQ & YL: 从来没有做过。
上课的时候TA给过 9 things can kill your PCR, 而且给了个简便的温度计算公式。
老板走的时候给我们的实验流程的反应温度跟按照简便公式计算出来的差距超级大……但是老板坚持，那些试验就是按照这个温度设计的！给我们三种细胞的cDNA（NIH-3T3, D3 cell line ES cell, a Mix differenating cell） 进行测试。
无语，不争。因为温度的话，有更复杂的公式可以算，但是差别不至于特别大。我自己也不明白，照做。
YL 跑去hall 的活动不来了。QQ结束了华文辩论会但是两个星期后要回国（现在走了）。我在上课。没有人可以全力以赴做这个实验。时间是挤的。没有指导。
两个星期，试了N次，QQ的手戴塑胶手套都过敏了。没结果就是没结果。反应用的reagent, cDNA, primer, 温度……修改了好多次。没结果……
摘抄我一周前的blog...
Run the PCR for whole week and did not get any result.
It did not show any band!!!
The cell line we use is mouse ES cell, D3 cell line, that is a high passage number cell line.
It was cultured in LIF medium to prevent its differenciation.
Then we did RT-PCR.
In RT, after using random hex as primer, we obtained 30ng/ul cDNA
Then used 17 different primers to test, inculding Fgf8, Bmp4, Wnt3a which we were interested for further study.
In the pcr cycle , we tried anneling temp at 58/57/56/50
Only oct-4, nanog showed bands at 58, sox2 showed multiple bands at 56, most of the rest did not show bands after PCR. 18S showed bands at 58 57 and 56.
Crying....
Our superviser ( oversea now) said most of the primers would not show in mES, but based on a paper we found, it did say that primers, such as Bmp4 shows!
老板的信里说，NIH-3T3一定会有东西，但是什么也没有。
最后，给实验室里另外一个人拿了另外一种细胞的cDNA, MC-3T3.自己偷改了反应温度,调低了3度， 结果终于出来了。
两个星期的辛苦，终于有了结果。就不明白，老板怎么要我们测试不会有结果的cDNA? 怎么给我们一个不合适的实验protocal? 虽然提出异议了但是他却说按照那上面的做.
Group member情况：
我，正在上LSM2202,学了PCR的一点皮毛。
QQ & YL: 从来没有做过。
上课的时候TA给过 9 things can kill your PCR, 而且给了个简便的温度计算公式。
老板走的时候给我们的实验流程的反应温度跟按照简便公式计算出来的差距超级大……但是老板坚持，那些试验就是按照这个温度设计的！给我们三种细胞的cDNA（NIH-3T3, D3 cell line ES cell, a Mix differenating cell） 进行测试。
无语，不争。因为温度的话，有更复杂的公式可以算，但是差别不至于特别大。我自己也不明白，照做。
YL 跑去hall 的活动不来了。QQ结束了华文辩论会但是两个星期后要回国（现在走了）。我在上课。没有人可以全力以赴做这个实验。时间是挤的。没有指导。
两个星期，试了N次，QQ的手戴塑胶手套都过敏了。没结果就是没结果。反应用的reagent, cDNA, primer, 温度……修改了好多次。没结果……
摘抄我一周前的blog...
Run the PCR for whole week and did not get any result.
It did not show any band!!!
The cell line we use is mouse ES cell, D3 cell line, that is a high passage number cell line.
It was cultured in LIF medium to prevent its differenciation.
Then we did RT-PCR.
In RT, after using random hex as primer, we obtained 30ng/ul cDNA
Then used 17 different primers to test, inculding Fgf8, Bmp4, Wnt3a which we were interested for further study.
In the pcr cycle , we tried anneling temp at 58/57/56/50
Only oct-4, nanog showed bands at 58, sox2 showed multiple bands at 56, most of the rest did not show bands after PCR. 18S showed bands at 58 57 and 56.
Crying....
Our superviser ( oversea now) said most of the primers would not show in mES, but based on a paper we found, it did say that primers, such as Bmp4 shows!
老板的信里说，NIH-3T3一定会有东西，但是什么也没有。
最后，给实验室里另外一个人拿了另外一种细胞的cDNA, MC-3T3.自己偷改了反应温度,调低了3度， 结果终于出来了。
两个星期的辛苦，终于有了结果。就不明白，老板怎么要我们测试不会有结果的cDNA? 怎么给我们一个不合适的实验protocal? 虽然提出异议了但是他却说按照那上面的做.
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