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how to clone plasmids

how to clone plasmids# Biology - 生物学

e*e2014-06-25 07:06

1 楼

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N*n2014-06-25 07:06

2 楼

【以下文字转载自 Chicago 讨论区】
发信人: xiaoxiaoren (可爱小宝宝), 信区: Chicago
标题: 2 hard pulls is nothing
发信站: BBS 未名空间站 (Sat Sep 29 21:55:06 2012, 美东)
If you don't want to buy a house with loan.
It is fairly true.
Ask yourself,
why do you need a such high credit score?

h*g2014-06-25 07:06

3 楼

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*e2014-06-25 07:06

4 楼

up. 收了一堆印度人的简历，没多少同胞的

N*n2014-06-25 07:06

5 楼

江山代有名媛出啊。。。。
芝加哥的小小名媛就想出个名，大家成全她吧。。。。

e*s2014-06-25 07:06

6 楼

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.

c*r2014-06-25 07:06

7 楼

肾一个就够了，要两个是多余的

h*g2014-06-25 07:06

8 楼

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

d*32014-06-25 07:06

9 楼

难道和果版名媛是两口子？

b*22014-06-25 07:06

10 楼

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】

b*l2014-06-25 07:06

11 楼

我有一点不明白：只有iphone区别对待？

【在 N********n 的大作中提到】

: 江山代有名媛出啊。。。。
: 芝加哥的小小名媛就想出个名，大家成全她吧。。。。

x*e2014-06-25 07:06

12 楼

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.

g*72014-06-25 07:06

13 楼

1 hard pull = $500

N*n2014-06-25 07:06

14 楼

ＢＡ　１０ｗ里程ｄｅａｌ还在的时候，申张卡轻松搞定$1200 cash 啊。。。

【在 g******7 的大作中提到】

: 1 hard pull = $500

N*n2014-06-25 07:06

15 楼

所以说名媛各有不同，这个组群偏执的性格独步天下。。。

d*02014-06-25 07:06

16 楼

这个是故事人的新马甲吧

N*n2014-06-25 07:06

17 楼

靠，ｓｔｏｒｙ９９８８您也知道？

【在 d*****0 的大作中提到】

: 这个是故事人的新马甲吧

m*w2014-06-25 07:06

18 楼

+1

【在 N********n 的大作中提到】

: 靠，ｓｔｏｒｙ９９８８您也知道？

N*n2014-06-25 07:06

19 楼

此小名媛与我印象最深的一次胡搅蛮缠就是其装b声称自己家里用别人送的Touchpad垫
桌角，我对其曰：牛啊。其就来劲了，大嘴巴开始胡吹，说这touchpad轻松去Amazon
trade-in 200多刀，我想，靠，32g的售价也就150，哪儿来的$2xx。
我当时手贱查了一下，HP Touchpad trade-in up to $85，小名媛这次牛b吹大了。我
就告诉她link，没想到其立马发了两个link给我，一个东芝pad，另外一个ipad 的
trade-in， up to $2xx, 我当场就笑喷了。。。。回个消息，告诉她我们讨论的是
touchpad，她来了一句：所有用手指点戳的，都是touch pad！！！
所有用手指点戳的，都是touch pad！！！
所有用手指点戳的，都是touch pad！！！
所有用手指点戳的，都是touch pad！！！

d*u2014-06-25 07:06

20 楼

靠，你连故事人都知道

【在 N********n 的大作中提到】

: 靠，ｓｔｏｒｙ９９８８您也知道？

a*n2014-06-25 07:06

21 楼

lol
i用的pad
都是ipad!

【在 N********n 的大作中提到】

: 此小名媛与我印象最深的一次胡搅蛮缠就是其装b声称自己家里用别人送的Touchpad垫
: 桌角，我对其曰：牛啊。其就来劲了，大嘴巴开始胡吹，说这touchpad轻松去Amazon
: trade-in 200多刀，我想，靠，32g的售价也就150，哪儿来的$2xx。
: 我当时手贱查了一下，HP Touchpad trade-in up to $85，小名媛这次牛b吹大了。我
: 就告诉她link，没想到其立马发了两个link给我，一个东芝pad，另外一个ipad 的
: trade-in， up to $2xx, 我当场就笑喷了。。。。回个消息，告诉她我们讨论的是
: touchpad，她来了一句：所有用手指点戳的，都是touch pad！！！
: 所有用手指点戳的，都是touch pad！！！
: 所有用手指点戳的，都是touch pad！！！
: 所有用手指点戳的，都是touch pad！！！