m********r 发帖数: 124 | 1 版上牛人多,特来问个问题:
PCR总是令人烦恼,国内的时候就遇到过,现在又出来折腾我了:
我是想分别扩e.coli两个蛋白的两个同源domain,都很小,200bp的样子,但总是得不到
,都只有引物二聚体。我不觉得是引物的问题,因为两组引物都失败了,而且以前没有
遇到过这个问题。我到觉得是模板的问题。我直接用的colony,然后用PCR的那个95度
10Min,理论上认为能裂菌。我在想是裂菌不充分呢?还是我开始加的菌落太多了?我
一直担心模板不够,挑个菌落在tube里twirl了很久。或者裂菌之后就是问题比较多,
我应该尝试的先制备chromosome再扩增呢?太头大了,牛人过来指点下吧 |
x********u 发帖数: 430 | 2 You can try alternative heating and cooling for breaking down the cell. 3X[
95C/1min and -80C/5min].
Try to use GoTaq Hot Start DNA polymerase.
If you still cannot get the desired band, try growing the cell in liquid
culture, and make genomic DNA by PureLink gDNA kit.
Since your fragment is around 200bp, why not just synthesize the sigle
strand oligoes and reconstitute your molecule by annealing on a PCR machine.
Good luck! |
d*p 发帖数: 534 | |
b******n 发帖数: 4225 | 4 agree
【在 d*p 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 菌量太多了反而做不出来的,用1/10试试看
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s********n 发帖数: 2939 | 5 如果还是不行,你可以试试这个protocol:
1. 挑一个colony加到5 ul solution 2 (plasmids prep);
2. pipette几次确保细胞裂解了(可以加热到100C for 2 min);
3. 加入0.5 ml 10 mM Tris-HCl (pH 8.0) or H2O;
4. 15,000xg for 5 min;
5. 用上清作为模板(0.5 or 1 ul);
6. 这个上清可以保存在-20C供下次再用。 |
m********r 发帖数: 124 | 6 我拿到e.coli的genome后,PCR就非常容易了,
克隆已经拿到,这周就做表达了,谢谢各位了
machine.
【在 x********u 的大作中提到】![](/moin_static193/solenoid/img/up.png) : You can try alternative heating and cooling for breaking down the cell. 3X[ : 95C/1min and -80C/5min]. : Try to use GoTaq Hot Start DNA polymerase. : If you still cannot get the desired band, try growing the cell in liquid : culture, and make genomic DNA by PureLink gDNA kit. : Since your fragment is around 200bp, why not just synthesize the sigle : strand oligoes and reconstitute your molecule by annealing on a PCR machine. : Good luck!
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