r******y
发帖数: 21907 | 1 是不是plasmid太长了，还是最好先到TA vector里再克隆到 gateway？ |
s**s
发帖数: 122 | 2 No problem if using Strategene site-directed mutagenesis kit.
Pick up couple of clones to confirm the protein expression or do sequencing.
【在 r******y 的大作中提到】
: 是不是plasmid太长了，还是最好先到TA vector里再克隆到 gateway？
|
r******y
发帖数: 21907 | 3 谢谢，是用的quickchange II，annealing 13 min，但是没有colony....
sequencing.
【在 s**s 的大作中提到】
: No problem if using Strategene site-directed mutagenesis kit.
: Pick up couple of clones to confirm the protein expression or do sequencing.
|
s**s
发帖数: 122 | 4 Increasing the amount of template to 150 ng might be helpful.
Increasing the annealing time to 15 min.
Also the quality of competent cells are important.
Good luck!
【在 r******y 的大作中提到】
: 谢谢，是用的quickchange II，annealing 13 min，但是没有colony....
:
: sequencing.
|
r******y
发帖数: 21907 | 5 thanks! it suggests 10 ng so i thought more might supress te reaction. will
try again! will give u baozi if i got colonies. :)
【在 s**s 的大作中提到】
: Increasing the amount of template to 150 ng might be helpful.
: Increasing the annealing time to 15 min.
: Also the quality of competent cells are important.
: Good luck!
|
f**u
发帖数: 346 | 6 recombineering可能更好
【在 r******y 的大作中提到】
: 是不是plasmid太长了，还是最好先到TA vector里再克隆到 gateway？
|
