how to add poly A sequence when making knock in vector?# Biology - 生物学
n*m
1 楼
交签证费要cgi号啥的, 怕父母搞不清, 所以想让外地的弟弟帮他们交签证费,签证后再
把护照寄到父母附近的中信银行. 这样交签证费和取护照的中信银行就不是同一个地方
了, 这样应该没有问题吧?
把护照寄到父母附近的中信银行. 这样交签证费和取护照的中信银行就不是同一个地方
了, 这样应该没有问题吧?
z*7
2 楼
我NIW的 PD 是 2010， Apr
今年夏天着急找工作就开始折腾EB1A， 12月140被RFE了，正在准备答复RFE中
今天一看NIW排期都到了 2010了，NIW只差了4个月，
我的情况是绿卡不急，但是工卡和旅游证急（我要找工作我老婆6月份要回国）
如果我转而等NIW了，不理EB1A RFE了，假如说下个月NIW能排到我的排期，从我申请工
卡，485 through NIW（not by EB1A)到最后拿到工卡（不是说绿卡）一般要多长时间
？？
是和EB1A一样长（2-3个月嘛）？还是说NIW 要比EB1A拿卡慢？
今年夏天着急找工作就开始折腾EB1A， 12月140被RFE了，正在准备答复RFE中
今天一看NIW排期都到了 2010了，NIW只差了4个月，
我的情况是绿卡不急，但是工卡和旅游证急（我要找工作我老婆6月份要回国）
如果我转而等NIW了，不理EB1A RFE了，假如说下个月NIW能排到我的排期，从我申请工
卡，485 through NIW（not by EB1A)到最后拿到工卡（不是说绿卡）一般要多长时间
？？
是和EB1A一样长（2-3个月嘛）？还是说NIW 要比EB1A拿卡慢？
j*i
3 楼
直接跟机箱盖板接触, 很容易实现被动散热.
为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
做被动散热
为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
做被动散热
y*u
4 楼
Anyone here have experience how to add polyA signal when making knock in
allelle?
someone said that we should not add any polyA signal at all by which they
assume that they are using the orginal polyA signal of the knock in allelle
allelle?
someone said that we should not add any polyA signal at all by which they
assume that they are using the orginal polyA signal of the knock in allelle
f*2
5 楼
没问题。
q*g
6 楼
Why dont you just link your 485 to your NIW assuming you filed 485
concurrently w/ EB1A and have the AP/EAD card in hands.
Any Da4 Niu2 explain the Pro/Con of interfiling 485 to older NIW application
.
concurrently w/ EB1A and have the AP/EAD card in hands.
Any Da4 Niu2 explain the Pro/Con of interfiling 485 to older NIW application
.
a*e
7 楼
太非主流了
CPU反装，然后通过heatpipe?这样背部会有个大空间
如果直接和背板接触，这个得保证两个板子的间隙，
定制还好，大规模够呛，背板spring load总是不靠谱
【在 j****i 的大作中提到】
: 直接跟机箱盖板接触, 很容易实现被动散热.
: 为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
: 做被动散热
CPU反装，然后通过heatpipe?这样背部会有个大空间
如果直接和背板接触，这个得保证两个板子的间隙，
定制还好，大规模够呛，背板spring load总是不靠谱
【在 j****i 的大作中提到】
: 直接跟机箱盖板接触, 很容易实现被动散热.
: 为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
: 做被动散热
b*o
8 楼
You don't have to add polyA to your cDNA
The knockin construct normally has its own promoter and polyA region.The expression cassette will be randomly inserted into (any) genomic DNA.
What's your mean "allele"?
When use "allele" it normally means "the genomic region of the gene", you can delete, mutate or add (tags etc) to any part of your allele in vitro, then knock it in.
allelle
【在 y*********u 的大作中提到】
: Anyone here have experience how to add polyA signal when making knock in
: allelle?
: someone said that we should not add any polyA signal at all by which they
: assume that they are using the orginal polyA signal of the knock in allelle
The knockin construct normally has its own promoter and polyA region.The expression cassette will be randomly inserted into (any) genomic DNA.
What's your mean "allele"?
When use "allele" it normally means "the genomic region of the gene", you can delete, mutate or add (tags etc) to any part of your allele in vitro, then knock it in.
allelle
【在 y*********u 的大作中提到】
: Anyone here have experience how to add polyA signal when making knock in
: allelle?
: someone said that we should not add any polyA signal at all by which they
: assume that they are using the orginal polyA signal of the knock in allelle
z*7
9 楼
thanks
你是说交一份EB1A485的钱，在假如说下个月NIW排期到的时候这个485可以转移到我的
NIW上？
这样link之后如果NIW（2010 Apr）在我Eb1A reply RFE deadline之前排期到了，我就
可以放弃回复Eb1A rfe了，用NIW申工卡就可以了
你是说交一份EB1A485的钱，在假如说下个月NIW排期到的时候这个485可以转移到我的
NIW上？
这样link之后如果NIW（2010 Apr）在我Eb1A reply RFE deadline之前排期到了，我就
可以放弃回复Eb1A rfe了，用NIW申工卡就可以了
b*u
10 楼
通过薄薄的机箱散，肯定效果差很远。不是越大越好。
【在 j****i 的大作中提到】
: 直接跟机箱盖板接触, 很容易实现被动散热.
: 为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
: 做被动散热
【在 j****i 的大作中提到】
: 直接跟机箱盖板接触, 很容易实现被动散热.
: 为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
: 做被动散热
y*u
11 楼
Thank you for your reply!
Sorry for not making this clear. By saying knock in I am referring to the
method utilizing homology recombination to replace an original gene allele
by another one pre designed.
What you are saying is usually refereed as 'transgenic event'.
expression cassette will be randomly inserted into (any) genomic DNA.
can delete, mutate or add (tags etc) to any part of your allele in vitro,
then knock it in.
【在 b*****o 的大作中提到】
: You don't have to add polyA to your cDNA
: The knockin construct normally has its own promoter and polyA region.The expression cassette will be randomly inserted into (any) genomic DNA.
: What's your mean "allele"?
: When use "allele" it normally means "the genomic region of the gene", you can delete, mutate or add (tags etc) to any part of your allele in vitro, then knock it in.
:
: allelle
Sorry for not making this clear. By saying knock in I am referring to the
method utilizing homology recombination to replace an original gene allele
by another one pre designed.
What you are saying is usually refereed as 'transgenic event'.
expression cassette will be randomly inserted into (any) genomic DNA.
can delete, mutate or add (tags etc) to any part of your allele in vitro,
then knock it in.
【在 b*****o 的大作中提到】
: You don't have to add polyA to your cDNA
: The knockin construct normally has its own promoter and polyA region.The expression cassette will be randomly inserted into (any) genomic DNA.
: What's your mean "allele"?
: When use "allele" it normally means "the genomic region of the gene", you can delete, mutate or add (tags etc) to any part of your allele in vitro, then knock it in.
:
: allelle
y*n
12 楼
需要机箱厂商在那一面加水冷。
【在 j****i 的大作中提到】
: 直接跟机箱盖板接触, 很容易实现被动散热.
: 为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
: 做被动散热
【在 j****i 的大作中提到】
: 直接跟机箱盖板接触, 很容易实现被动散热.
: 为啥没有主板厂商这么做. 主板提供两种选择, CPU在正面的方便用风冷, CPU在背面的
: 做被动散热
g*y
13 楼
1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
PCR adding those sequence right after the stop codon.
2. It all depends on how you design your KI allele. If your KI allele is
only to replace one exon, then you don't need to worry about anything. If
you are to insert a cDNA sequence right at the 1st exon,
then you may have to worry about non-sense mediated dacay. In this case, it
would be safer to add an exogenous pA.
allelle
【在 y*********u 的大作中提到】
: Anyone here have experience how to add polyA signal when making knock in
: allelle?
: someone said that we should not add any polyA signal at all by which they
: assume that they are using the orginal polyA signal of the knock in allelle
PCR adding those sequence right after the stop codon.
2. It all depends on how you design your KI allele. If your KI allele is
only to replace one exon, then you don't need to worry about anything. If
you are to insert a cDNA sequence right at the 1st exon,
then you may have to worry about non-sense mediated dacay. In this case, it
would be safer to add an exogenous pA.
allelle
【在 y*********u 的大作中提到】
: Anyone here have experience how to add polyA signal when making knock in
: allelle?
: someone said that we should not add any polyA signal at all by which they
: assume that they are using the orginal polyA signal of the knock in allelle
y*u
14 楼
Thank you! what I did is completely replaced the exon1, exon2, and intron1
Do you think we need to add an exogenous pA signal in this case?
I added it anyway
it
【在 g***y 的大作中提到】
: 1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
: PCR adding those sequence right after the stop codon.
: 2. It all depends on how you design your KI allele. If your KI allele is
: only to replace one exon, then you don't need to worry about anything. If
: you are to insert a cDNA sequence right at the 1st exon,
: then you may have to worry about non-sense mediated dacay. In this case, it
: would be safer to add an exogenous pA.
:
: allelle
Do you think we need to add an exogenous pA signal in this case?
I added it anyway
it
【在 g***y 的大作中提到】
: 1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
: PCR adding those sequence right after the stop codon.
: 2. It all depends on how you design your KI allele. If your KI allele is
: only to replace one exon, then you don't need to worry about anything. If
: you are to insert a cDNA sequence right at the 1st exon,
: then you may have to worry about non-sense mediated dacay. In this case, it
: would be safer to add an exogenous pA.
:
: allelle
y*u
15 楼
顶上去继续问！！
y*u
16 楼
We replaced the part of exon1,and exon2, do you think the endogenous pA
would work in this case? And could you post a link regarding the case of
utilizing the endogenous pA ?
it
【在 g***y 的大作中提到】
: 1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
: PCR adding those sequence right after the stop codon.
: 2. It all depends on how you design your KI allele. If your KI allele is
: only to replace one exon, then you don't need to worry about anything. If
: you are to insert a cDNA sequence right at the 1st exon,
: then you may have to worry about non-sense mediated dacay. In this case, it
: would be safer to add an exogenous pA.
:
: allelle
would work in this case? And could you post a link regarding the case of
utilizing the endogenous pA ?
it
【在 g***y 的大作中提到】
: 1. A lot of expression vectors contain SV40 pA or BGH pA. You can simply
: PCR adding those sequence right after the stop codon.
: 2. It all depends on how you design your KI allele. If your KI allele is
: only to replace one exon, then you don't need to worry about anything. If
: you are to insert a cDNA sequence right at the 1st exon,
: then you may have to worry about non-sense mediated dacay. In this case, it
: would be safer to add an exogenous pA.
:
: allelle
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