X*n
3 楼
应该很容易。
g*5
4 楼
sunny in joke ban, no here :)
X*n
5 楼
如果你不关注酵母background的话,可以直接买一个knockout菌株,把你的基因克隆到
一个酵母表达载体(pRS416什么的)里面就可以转化了。
一个酵母表达载体(pRS416什么的)里面就可以转化了。
g*5
7 楼
谢谢大家。看到thermo 有现成的敲除的strain卖,就是基因型看得头昏。
Strain Background Genotype
Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
Het/Hom Dip BY4743 4741/4742
我要的菌株是Yeast HetDip Knock Out Strain YJR045C
怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
Strain Background Genotype
Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
Het/Hom Dip BY4743 4741/4742
我要的菌株是Yeast HetDip Knock Out Strain YJR045C
怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
T*i
8 楼
你要先看 yeast KO 的表型,SGD查一下。如果lethal,只好买het,否则买MATa 或
MATalpha都行。Complementation test 的时候也是一样。如果是lethal,那你需要做
plasmid shuffling/exchange;如果不是lethal,那就好办,直接转目标plasmid 就可
以了。
【在 g*********5 的大作中提到】![](/moin_static193/solenoid/img/up.png)
: 谢谢大家。看到thermo 有现成的敲除的strain卖,就是基因型看得头昏。
: Strain Background Genotype
: Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
: Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
: Het/Hom Dip BY4743 4741/4742
: 我要的菌株是Yeast HetDip Knock Out Strain YJR045C
: 怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
MATalpha都行。Complementation test 的时候也是一样。如果是lethal,那你需要做
plasmid shuffling/exchange;如果不是lethal,那就好办,直接转目标plasmid 就可
以了。
【在 g*********5 的大作中提到】
![](/moin_static193/solenoid/img/up.png)
: 谢谢大家。看到thermo 有现成的敲除的strain卖,就是基因型看得头昏。
: Strain Background Genotype
: Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
: Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
: Het/Hom Dip BY4743 4741/4742
: 我要的菌株是Yeast HetDip Knock Out Strain YJR045C
: 怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
T*i
9 楼
补充一下,如果是lethal,het需要先转那个control plasmid (with the yeast
ortholog of your mammalian target) 再sporulate。还有别的策略,一时说不清,最
好周围找个yeast 专家问问。不是lethal,怎么都好办。
【在 g*********5 的大作中提到】![](/moin_static193/solenoid/img/up.png)
: 谢谢大家。看到thermo 有现成的敲除的strain卖,就是基因型看得头昏。
: Strain Background Genotype
: Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
: Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
: Het/Hom Dip BY4743 4741/4742
: 我要的菌株是Yeast HetDip Knock Out Strain YJR045C
: 怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
ortholog of your mammalian target) 再sporulate。还有别的策略,一时说不清,最
好周围找个yeast 专家问问。不是lethal,怎么都好办。
【在 g*********5 的大作中提到】
![](/moin_static193/solenoid/img/up.png)
: 谢谢大家。看到thermo 有现成的敲除的strain卖,就是基因型看得头昏。
: Strain Background Genotype
: Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
: Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
: Het/Hom Dip BY4743 4741/4742
: 我要的菌株是Yeast HetDip Knock Out Strain YJR045C
: 怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
g*5
12 楼
This gene is a mammalian gene, I have the human gene at hand.
X*n
17 楼
你的基因是个essential gene。 你买的这个是heterzygous deletion,很多时候这种
deletion没有表型。所以你应该先看看有没有你想要的表型,然后才做互补试验。
如果没有表型,你可以参考上面Tianzi的回复所说的方法,也可以去找找有没有ts
mutant。或者去看看Yeast DAmp Library 或者 TR Hughes 的 TetO7-promoter
mutants library有没有你想要的基因。
deletion没有表型。所以你应该先看看有没有你想要的表型,然后才做互补试验。
如果没有表型,你可以参考上面Tianzi的回复所说的方法,也可以去找找有没有ts
mutant。或者去看看Yeast DAmp Library 或者 TR Hughes 的 TetO7-promoter
mutants library有没有你想要的基因。
g*5
18 楼
this gene is not in TR Hughes library but in Damp library.
For Damp library,
haploid or heterozygous diploid formats
which one is better for the experiment?
thanks, baozi.
【在 X******n 的大作中提到】![](/moin_static193/solenoid/img/up.png)
: 你的基因是个essential gene。 你买的这个是heterzygous deletion,很多时候这种
: deletion没有表型。所以你应该先看看有没有你想要的表型,然后才做互补试验。
: 如果没有表型,你可以参考上面Tianzi的回复所说的方法,也可以去找找有没有ts
: mutant。或者去看看Yeast DAmp Library 或者 TR Hughes 的 TetO7-promoter
: mutants library有没有你想要的基因。
For Damp library,
haploid or heterozygous diploid formats
which one is better for the experiment?
thanks, baozi.
【在 X******n 的大作中提到】
![](/moin_static193/solenoid/img/up.png)
: 你的基因是个essential gene。 你买的这个是heterzygous deletion,很多时候这种
: deletion没有表型。所以你应该先看看有没有你想要的表型,然后才做互补试验。
: 如果没有表型,你可以参考上面Tianzi的回复所说的方法,也可以去找找有没有ts
: mutant。或者去看看Yeast DAmp Library 或者 TR Hughes 的 TetO7-promoter
: mutants library有没有你想要的基因。
l*1
20 楼
pRS416 excepts T7 also has Plac promoter.
pls refer
PMID 16820502
http://www.ncbi.nlm.nih.gov/pubmed/16820502
>pRS416 is a centromeric (CEN) plasmid and therefore is maintained at 1 to 2
copies per cell, while pRS426 is a 2μm plasmid, maintained at about 25
copies per cell (36). Both plasmids have the ColE1-derived pBluescript high-
copy origin of replication, a lacZα-associated multicloning site, an
ampicillin resistance gene (bla) for gram-negative bacteria, and a URA3
marker for yeast. These plasmids also have T7 and Plac promoters that can be
utilized for gene expression.
or
http://aem.asm.org/content/72/7/5027.full
Ps: original hint was from
wurt
2012-02-04 03:30:25
来自: 202.
9f
>The streaking do not 'dilute' the plasmid, but simply purify the colonies.
The copy number of the plasmid mostly depend on your replication origin (
never use 2u origin if you want a low copy), and whether the plasmid contain
centromere (like CEN6 on pRS416). Usually the centromere sequence can limit
the copy number of your plasmid.
I don't think you need to wash or replica your plates. If you don't want to
have too many plasmids in one cell, just simply reduce the transformation
efficiency and
http://www.google.co.uk/#q=PRS416+promoter+未名+存档
please only google likes above,
i mean only limited to mitbbs Bio/medi Ban former post
then at least you can know knew key word which focuses on your target.
>
发信人: giantbird05 (大鸟), 信区: Biology
标 题: Re: 酵母互补试验好做吗?
发信站: BBS 未名空间站 (Fri Oct 11 00:12:03 2013, 美东)
请问pRS416是什么启动子阿?
谢谢。
>>
>>>
【在 g*********5 的大作中提到】![](/moin_static193/solenoid/img/up.png)
: pRS416 mcs ?
: under the T7 ?
: would you please give me a link?
: I googled and more confused.
: thanks.
pls refer
PMID 16820502
http://www.ncbi.nlm.nih.gov/pubmed/16820502
>pRS416 is a centromeric (CEN) plasmid and therefore is maintained at 1 to 2
copies per cell, while pRS426 is a 2μm plasmid, maintained at about 25
copies per cell (36). Both plasmids have the ColE1-derived pBluescript high-
copy origin of replication, a lacZα-associated multicloning site, an
ampicillin resistance gene (bla) for gram-negative bacteria, and a URA3
marker for yeast. These plasmids also have T7 and Plac promoters that can be
utilized for gene expression.
or
http://aem.asm.org/content/72/7/5027.full
Ps: original hint was from
wurt
2012-02-04 03:30:25
来自: 202.
9f
>The streaking do not 'dilute' the plasmid, but simply purify the colonies.
The copy number of the plasmid mostly depend on your replication origin (
never use 2u origin if you want a low copy), and whether the plasmid contain
centromere (like CEN6 on pRS416). Usually the centromere sequence can limit
the copy number of your plasmid.
I don't think you need to wash or replica your plates. If you don't want to
have too many plasmids in one cell, just simply reduce the transformation
efficiency and
http://www.google.co.uk/#q=PRS416+promoter+未名+存档
please only google likes above,
i mean only limited to mitbbs Bio/medi Ban former post
then at least you can know knew key word which focuses on your target.
>
发信人: giantbird05 (大鸟), 信区: Biology
标 题: Re: 酵母互补试验好做吗?
发信站: BBS 未名空间站 (Fri Oct 11 00:12:03 2013, 美东)
请问pRS416是什么启动子阿?
谢谢。
>>
>>>
【在 g*********5 的大作中提到】
![](/moin_static193/solenoid/img/up.png)
: pRS416 mcs ?
: under the T7 ?
: would you please give me a link?
: I googled and more confused.
: thanks.
g*5
21 楼
what different between Mat A and Mat Alpha?
one haploid one diploid?
【在 g*********5 的大作中提到】![](/moin_static193/solenoid/img/up.png)
: 谢谢大家。看到thermo 有现成的敲除的strain卖,就是基因型看得头昏。
: Strain Background Genotype
: Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
: Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
: Het/Hom Dip BY4743 4741/4742
: 我要的菌株是Yeast HetDip Knock Out Strain YJR045C
: 怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
one haploid one diploid?
【在 g*********5 的大作中提到】
![](/moin_static193/solenoid/img/up.png)
: 谢谢大家。看到thermo 有现成的敲除的strain卖,就是基因型看得头昏。
: Strain Background Genotype
: Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
: Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
: Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
: Het/Hom Dip BY4743 4741/4742
: 我要的菌株是Yeast HetDip Knock Out Strain YJR045C
: 怎么样的平板才能使敲除的菌株不长啊?能买到板吗?
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