b**x
发帖数: 8 | 1 I want to assemble a gene from three PCR fragments into one plasmid.
Clonetech 有个kit，试用了但从未成功过。望不吝赐教。 |
j****x
发帖数: 1704 | 2 设计好酶切位点几个片段丢一起直接连接就行了，和普通连接一样没啥可讲究的，关键
是处理好每个PCR产物，酶切要尽可能彻底。
实在不行就一个片段一个片段的连接，其实相比起来也耽误不了几天时间 |
n********k
发帖数: 2818 | 3 for not very big fragments, what the 2L said works well with 3 fragments or even
more, I never tried more than 4 though. For big fragments, could be harder..
.alternatively, fusion PCR, many of this kind of cloning can be done in a
single fusion-PCR reaction...
【在 b**x 的大作中提到】
: I want to assemble a gene from three PCR fragments into one plasmid.
: Clonetech 有个kit，试用了但从未成功过。望不吝赐教。
|
s********n
发帖数: 2939 | |
b**x
发帖数: 8 | 5 I have seen publications about sLIC. Did it work as well
as advertised? The Clonetech kit was a huge disappointment
even though their Ads made it sound so easy.
【在 s********n 的大作中提到】
: sLIC.
|
s********n
发帖数: 2939 | 6 If you could gel purify the fragments, the efficiency is really high. We use
this technique a lot here. |
b**x
发帖数: 8 | 7 Thanks a lot, SmartKevin.
Did you follow the 2007 Nature Biotech paper by Elledge or some other
paper? Did you use RecA- strain or regular strains? If you have a protocol
to spare, 站内通信。
use
【在 s********n 的大作中提到】
: If you could gel purify the fragments, the efficiency is really high. We use
: this technique a lot here.
|
s********n
发帖数: 2939 | 8 I usually use LIC to clone genes (single fragments). However, I know other
people here use the same protocol as you mentioned. Gel-purification is key
for sLIC.
【在 b**x 的大作中提到】
: Thanks a lot, SmartKevin.
: Did you follow the 2007 Nature Biotech paper by Elledge or some other
: paper? Did you use RecA- strain or regular strains? If you have a protocol
: to spare, 站内通信。
:
: use
|
