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some question about cloning,thank you!

some question about cloning,thank you!# Biology - 生物学

D*32013-04-08 07:04

1 楼

Refurbished: DELL Latitude E5410 Intel Core i5 2.60GHz 4GB Memory 160GB HDD
14.1" Notebook Windows 7 Professional 64-bit
Intel Core i5 2.60GHz
4GB Memory 160GB HDD
Windows 7 Professional 64-bit
$239.99
价格貌似不错
有更好的i5 notebook DEAL么？

m*32013-04-08 07:04

2 楼

I want to clone some genes into the pET30a vector and the p
GBKT7 vector which both contain an N-terminus tag,do I have to exclude the
ATG in my own gene?

s*d2013-04-08 07:04

3 楼

哪里的deal，价格很不错啊

s*y2013-04-08 07:04

4 楼

如果留着你的ATG的话，会有一定比例跳过tag 而在你的ATG 开始。
这个看你干什么用，如果是提纯蛋白的话无所谓，因为没有tag 的最后就不被提纯了。

【在 m*******3 的大作中提到】

: I want to clone some genes into the pET30a vector and the p
: GBKT7 vector which both contain an N-terminus tag,do I have to exclude the
: ATG in my own gene?

D*32013-04-08 07:04

5 楼

newegg

【在 s*******d 的大作中提到】

: 哪里的deal，价格很不错啊

m*32013-04-08 07:04

6 楼

谢谢艳阳天老师！

【在 s******y 的大作中提到】

: 如果留着你的ATG的话，会有一定比例跳过tag 而在你的ATG 开始。
: 这个看你干什么用，如果是提纯蛋白的话无所谓，因为没有tag 的最后就不被提纯了。

b*n2013-04-08 07:04

7 楼

That's probably first generation i5, which is easily overheated specially
when it gets old.

【在 D***3 的大作中提到】

: newegg

g*52013-04-08 07:04

8 楼

不需要。

D*32013-04-08 07:04

9 楼

200刀淘了个

H*i2013-04-08 07:04

10 楼

往上数6个nt 然后看看附近有没有AGGA类似的序列，没有得话基本不用担心这个，没有
RBS得话比例太低了。。
另外pet系列在NcoI酶切位点有一个ATG,LZ愿意的话可以用这个。

【在 s******y 的大作中提到】

: 如果留着你的ATG的话，会有一定比例跳过tag 而在你的ATG 开始。
: 这个看你干什么用，如果是提纯蛋白的话无所谓，因为没有tag 的最后就不被提纯了。

m*32013-04-08 07:04

11 楼

Nco1往上没有AGGA，那就不会从这儿起始是吧？

【在 H*******i 的大作中提到】

: 往上数6个nt 然后看看附近有没有AGGA类似的序列，没有得话基本不用担心这个，没有
: RBS得话比例太低了。。
: 另外pet系列在NcoI酶切位点有一个ATG,LZ愿意的话可以用这个。

l*12013-04-08 07:04

12 楼

RE LZ
also 'ACGA' pls check if with or withnot
pls refer
The ribosome binding site (RBS; also known as the shine-delgarno sequence).
This is a series of nucleotides located 7-13 bases upstream of the start
codon. This sequence is recognized by the ribosome (the protein complex that
translates mRNA sequence information into protein).
from the bacteria E. coli and is:
AGGAGGA
In order to select a start codon you need to do two things;
1. Look at the various options for start codons. For each one, look upstream
(to the left) about 7-13 nucleotides (nts) and see if you can see something
that looks similar to the consensus RBS (AGGAGGA).
GTA-'ACGAGGA'-TAATGGAATG
For this example I have colored the start codon green and the RBS blue. You
can see that this is not an exact match (since the second letter is C and
not G) but it is very close. Not all will be this close but many are. As
with everything else, if you are not sure, don't change anything.
more pls go to web site:
http://depts.washington.edu/agro/genomes/students/stanstart.htm
Btw,
HaibaraAi means that after Nco I 酶切 , that 位点ATG will be cut and can'
t be start codon after Nco I 酶切...
>

>>

【在 m*******3 的大作中提到】

: Nco1往上没有AGGA，那就不会从这儿起始是吧？