u*********1
发帖数: 2518 | 1 正在制作用于whole-genome sequencing的human genome library。做完ligation跑胶
看到smear，然后切下比如450-500bp的区间做PCR。但可能是50bp的interval太小或者
自己技术不好，所以最后PCR出来的亮度比较低。（当然也不排除ligation efficiency
不好）
PCR的结果如图。
还没拿去测浓度。只想问问有经验的前辈这样的情况能拿去测30 coverage的WGS吗？
另外还有一个问题，就是，我们切胶的smear每一个区间（300-400，400-500，500-600
）这些区间里都一定cover了genome的每一个部分吗？我总担心genome的有的部分没有
被涵盖到，那岂不是就人为引入了很多bias？
thx |
y******8
发帖数: 1764 | 2 If you can recover 10 ng DNA after gel cut, it should be enough.
Your gel picture looks weird. The band near to 1kb is too strong to me.
You should have a more quantitative way to measure your DNA concentration
around 1ng/ul. |
u*********1
发帖数: 2518 | 3 Thanks
But I just didn't see any band around 1kb....you mean 100bp?
【在 y******8 的大作中提到】
: If you can recover 10 ng DNA after gel cut, it should be enough.
: Your gel picture looks weird. The band near to 1kb is too strong to me.
: You should have a more quantitative way to measure your DNA concentration
: around 1ng/ul.
|
y******8
发帖数: 1764 | 4 I saw two bands. What molecular weight should they be? |
o********r
发帖数: 775 | 5 30X一般是平均，具体到单个base，貌似现在publication的标准是80%以上（base or
exon）在20X以上。30X总还有不少base是没有任何coverage的。还有不少重要基因的
coverage总是比较低，比如NOTCH1 |
