z*********8
发帖数: 1203 | 1 用过的达人请指点，我需要把3段DNA按照一定顺序克隆到plasmid上面，但是好用的
resctriciton enzyme在3段DNA里都有，所以ligation based cloning不太能用。然后
别人就介绍了这个infusion kit，发现克隆1个基因好用，克隆3个基因总是中间的那个
基因在，旁边的都不在，不知道有没有什么trick好克隆？谢谢大家！ |
z*********8
发帖数: 1203 | 2 以前用的都是overlap pcr，都还不错，3段一般还可以接起来。做fusion protein,
protein tag，mutagenesis之类的还不错。最近做的这个比较棘手，PCR不出来了，只
有依靠infusion了。问题是，我按照clonetech人家网站上的一步步做下来，也用了他
家的primer design tool了，还是不行，只有中间一段DNA在上面，2端的DNA没有。大
家快快出招！谢谢！ |
N2
发帖数: 81 | 3 It should work. This kind of method is very efficient. Here are some points
maybe be useful.
1) Gel purify you PCR product.
2)use Nanodrop to estimate the concentration of your PCR products
3) add all fragment 1:1 ratio to vector. Add 100-200ng vector part. It also
depends your fragment size. you may add up to 500ug total DNA per reaction I
guess.
4) you can put 20-40nt overlap between joint points.
How do you screen you positive colonies? Use cloning PCR to check the joints
? you can just screen more colonies.
5)check you DNA sequence carefully to see if there structure weird. Or some
sequence can cause toxicity.
Good luck! |
z*********8
发帖数: 1203 | 4 我别的都跟你说的那样做的，只有pcr我跑胶看到都是单条带，所以就只有 column
purification。你觉得是我没有割胶纯化的问题么？？
发信人: N2 (Huahua), 信区: Biology
标 题: Re: 怎样用infusion kit克隆多个PCR 片段？？
发信站: BBS 未名空间站 (Tue Jul 26 17:52:12 2011, 美东)
It should work. This kind of method is very efficient. Here are some points
maybe be useful.
1) Gel purify you PCR product.
2)use Nanodrop to estimate the concentration of your PCR products
3) add all fragment 1:1 ratio to vector. Add 100-200ng vector part. It also
depends your fragment size. you may add up to 500ug total DNA per reaction I
guess.
4) you can put 20-40nt overlap between joint points.
How do you screen you positive colonies? Use cloning PCR to check the joints
? you can just screen more colonies.
5)check you DNA sequence carefully to see if there structure weird. Or some
sequence can cause toxicity.
Good luck! |
N2
发帖数: 81 | 5 It is really hard to say yes or no.
Most of time, if you do just column purification, you got low ligation
efficiency because the primers in it.
it always depends specific case. For some hard ligation, you need to be
careful for every steps.
There are many reasons to fail a ligation. you may just meet a hard bone.
You can drop me a mail with the vector and inserts sequences, your primers.
I can help to have a look.
points
also
【在 z*********8 的大作中提到】
: 我别的都跟你说的那样做的，只有pcr我跑胶看到都是单条带，所以就只有 column
: purification。你觉得是我没有割胶纯化的问题么？？
: 发信人: N2 (Huahua), 信区: Biology
: 标 题: Re: 怎样用infusion kit克隆多个PCR 片段？？
: 发信站: BBS 未名空间站 (Tue Jul 26 17:52:12 2011, 美东)
: It should work. This kind of method is very efficient. Here are some points
: maybe be useful.
: 1) Gel purify you PCR product.
: 2)use Nanodrop to estimate the concentration of your PCR products
: 3) add all fragment 1:1 ratio to vector. Add 100-200ng vector part. It also
|
z*********8
发帖数: 1203 | |
c******g
发帖数: 49 | 7
points
also
column purification会把几十bp的primers一起纯化出来，尽管有时候gel上就只看到
一条带，所以我一般都是gel purification。
【在 z*********8 的大作中提到】
: 我别的都跟你说的那样做的，只有pcr我跑胶看到都是单条带，所以就只有 column
: purification。你觉得是我没有割胶纯化的问题么？？
: 发信人: N2 (Huahua), 信区: Biology
: 标 题: Re: 怎样用infusion kit克隆多个PCR 片段？？
: 发信站: BBS 未名空间站 (Tue Jul 26 17:52:12 2011, 美东)
: It should work. This kind of method is very efficient. Here are some points
: maybe be useful.
: 1) Gel purify you PCR product.
: 2)use Nanodrop to estimate the concentration of your PCR products
: 3) add all fragment 1:1 ratio to vector. Add 100-200ng vector part. It also
|
N2
发帖数: 81 | 8 Cong!
【在 z*********8 的大作中提到】
: 谢谢建议，通过割胶纯化又做了一次，成功了！
|
z*********8
发帖数: 1203 | |
