h*********g
发帖数: 18 | 1 I got several plasmids from other lab and need to clone them and then do
maxiprep.
We have TOP10F' E.coli.
I will add 2 ul plasmid (100ng/ul) into one tube of E.coli TOP10F', mix, put
one ice for 5-30 min. Then heat-shock for 1 min at 42 degree.
Transfer the tube on ice. Add 250 ul room temperature SOC medium. shake the
tube at 37 for 1 hour.
Then spread 10-50ul onto a plate.
Is that right? I am new to cloning. Please help. Thanks. |
e****s
发帖数: 1125 | 2 This is transformation not cloning.
Looks about right. Just think 200 ng is too much. 20-50 ng is enough.
put
the
【在 h*********g 的大作中提到】
: I got several plasmids from other lab and need to clone them and then do
: maxiprep.
: We have TOP10F' E.coli.
: I will add 2 ul plasmid (100ng/ul) into one tube of E.coli TOP10F', mix, put
: one ice for 5-30 min. Then heat-shock for 1 min at 42 degree.
: Transfer the tube on ice. Add 250 ul room temperature SOC medium. shake the
: tube at 37 for 1 hour.
: Then spread 10-50ul onto a plate.
: Is that right? I am new to cloning. Please help. Thanks.
|
h*********g
发帖数: 18 | 3 Yes. It is transformation. After they grow on a plate, I will pick one
colony to grow it in the liquid culture medium. Thanks.
【在 e****s 的大作中提到】
: This is transformation not cloning.
: Looks about right. Just think 200 ng is too much. 20-50 ng is enough.
:
: put
: the
|
b***2
发帖数: 348 | 4 Make sure your bacteria are competent cells and still maintain their
competency. Follow video on youtube if it is the first time for you to do
transformation.
put
the
【在 h*********g 的大作中提到】
: I got several plasmids from other lab and need to clone them and then do
: maxiprep.
: We have TOP10F' E.coli.
: I will add 2 ul plasmid (100ng/ul) into one tube of E.coli TOP10F', mix, put
: one ice for 5-30 min. Then heat-shock for 1 min at 42 degree.
: Transfer the tube on ice. Add 250 ul room temperature SOC medium. shake the
: tube at 37 for 1 hour.
: Then spread 10-50ul onto a plate.
: Is that right? I am new to cloning. Please help. Thanks.
|
x********e
发帖数: 35261 | 5 做大提之前最好酶切鉴定加测序关键位置
外来的质粒最好都double check一下
【在 h*********g 的大作中提到】
: Yes. It is transformation. After they grow on a plate, I will pick one
: colony to grow it in the liquid culture medium. Thanks.
|
