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】![](/moin_static193/solenoid/img/up.png) : 是不是plasmid太长了,还是最好先到TA vector里再克隆到 gateway?
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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】![](/moin_static193/solenoid/img/up.png) : No problem if using Strategene site-directed mutagenesis kit. : Pick up couple of clones to confirm the protein expression or do sequencing.
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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】![](/moin_static193/solenoid/img/up.png) : 谢谢,是用的quickchange II,annealing 13 min,但是没有colony.... : : sequencing.
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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】![](/moin_static193/solenoid/img/up.png) : 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!
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f**u 发帖数: 346 | 6 recombineering可能更好
【在 r******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 是不是plasmid太长了,还是最好先到TA vector里再克隆到 gateway?
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