j******n
发帖数: 941 | 1 用的Origene pCMV6-Entry的骨架
发现这个骨架本身用Zymo的z competent转化效率就特别低 怀疑是低拷贝质粒
5ul质粒+50ulcompetent也只能长出十来个克隆
可是用这个骨架酶切后与目的基因PCR酶切产物做T4连接后更死活长不出克隆来
请问大家有啥建议？换competent？哪个公司的DH5a转化效率更好些？ | b******n
发帖数: 4225 | 2 invitrogen
号称10^9
如果还不行就用电转吧，呜泱呜泱的一大片
【在 j******n 的大作中提到】
: 用的Origene pCMV6-Entry的骨架
: 发现这个骨架本身用Zymo的z competent转化效率就特别低 怀疑是低拷贝质粒
: 5ul质粒+50ulcompetent也只能长出十来个克隆
: 可是用这个骨架酶切后与目的基因PCR酶切产物做T4连接后更死活长不出克隆来
: 请问大家有啥建议？换competent？哪个公司的DH5a转化效率更好些？
| r****d
发帖数: 4 | 3 pEntry is a high copy number plasmid. I knew the person who constructed this
vector and had cloned a lot gene into it. I never had any problem. First,
what is the concentration of Kan for your plate, I usually use 20 to 30 mg/l
. Second, the volume of plasmid should be less than 5% of total volume of E.
coli. You can try the competent cell from bioline. For PCR ligation, if you
do a 12 degree over night ligation, the efficiency is better. | j******n
发帖数: 941 | 4
this
/l
E.
you
谢谢你的建议 确实kan的浓度非常影响这个质粒 我曾经用50ug/ml 一个克隆也出不来
后来按protocol降到25 就能出来十多个了我回头试试低温过夜看看
【在 r****d 的大作中提到】
: pEntry is a high copy number plasmid. I knew the person who constructed this
: vector and had cloned a lot gene into it. I never had any problem. First,
: what is the concentration of Kan for your plate, I usually use 20 to 30 mg/l
: . Second, the volume of plasmid should be less than 5% of total volume of E.
: coli. You can try the competent cell from bioline. For PCR ligation, if you
: do a 12 degree over night ligation, the efficiency is better.
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