r**h
发帖数: 6 | 1 请教有关专家：
在Sanger没有找到该位点的129的BAC clone，还有什么library可以推荐?比如说RPCI-
22怎么样？不过我没找到定位single clone的bioinformatics工具，比如ensembl之类。
不准备自己做hybridization to screen the clone，因为花费力气和金钱比较多；
也不希望直接打C2 ES cell line，担心效率会有所下降；
也不知道在该位点，129和C57的区别会有多大，不知道用C57的BAC做的construct打129
ES cell，会不会比较不好；
所以回到开始的问题：如果执着于129 BAC clone，除了Sanger，还有什么library选择
（如果不自己筛的话）？或者说，应该执着于129 BAC吗？
完全新手一个，简单问题比较多，贻笑大方了。谢谢！ | g***y
发帖数: 201 | 2 多年没做这个了，知识比较老化。
1.比较两个arm的sequence homology between 129 and B6.
一般的担忧是因为两个strain的序列有差别，可能影响同源重组的效率。但是如果幸运
的话也许你感兴趣的区域两者高度同源。
否则，几个OPTIONs
A。选用B6 derived ES CELL,有些service提供的。
B.让你的arm尽量长. 但是还是不能保证一定会有positive clone,要看运气，并且问题
是plasmid大到一定程度就不稳定了。比较safe的办法是用BAC recombineering做
BAC based construct.
C。 use long-range high fidelity DNA polymerase to PCR 129 genomic sequence.
这个对cloning要求有点高，并且要做sequencing确定没PCR error
【在 r**h 的大作中提到】
: 请教有关专家：
: 在Sanger没有找到该位点的129的BAC clone，还有什么library可以推荐?比如说RPCI-
: 22怎么样？不过我没找到定位single clone的bioinformatics工具，比如ensembl之类。
: 不准备自己做hybridization to screen the clone，因为花费力气和金钱比较多；
: 也不希望直接打C2 ES cell line，担心效率会有所下降；
: 也不知道在该位点，129和C57的区别会有多大，不知道用C57的BAC做的construct打129
: ES cell，会不会比较不好；
: 所以回到开始的问题：如果执着于129 BAC clone，除了Sanger，还有什么library选择
: （如果不自己筛的话）？或者说，应该执着于129 BAC吗？
: 完全新手一个，简单问题比较多，贻笑大方了。谢谢！
| r**h
发帖数: 6 | 3 谢谢指教。
找不到129的BAC，也许意味着129的这个位点没有测序，所以看来比较难比较两个
strain的序列。我自己也许可以PCR出来，自己测，不过感觉事倍功半。我想当然的比
较多，还请指正。
比较遗憾我们使用的service质量不算上乘，做B6 derived，有点战战兢兢。LOL...
谢谢指出这一点。我准备用recombineering，所以希望有BAC。但是如果找不到，就只
能PCR 129 arms，然后一步步酶切连接了。
sequence.
非常感谢你的答复。
【在 g***y 的大作中提到】
: 多年没做这个了，知识比较老化。
: 1.比较两个arm的sequence homology between 129 and B6.
: 一般的担忧是因为两个strain的序列有差别，可能影响同源重组的效率。但是如果幸运
: 的话也许你感兴趣的区域两者高度同源。
: 否则，几个OPTIONs
: A。选用B6 derived ES CELL,有些service提供的。
: B.让你的arm尽量长. 但是还是不能保证一定会有positive clone,要看运气，并且问题
: 是plasmid大到一定程度就不稳定了。比较safe的办法是用BAC recombineering做
: BAC based construct.
: C。 use long-range high fidelity DNA polymerase to PCR 129 genomic sequence.
| n********k
发帖数: 2818 | 4 All good suggestions...I would vote for the second one as many core facility
prefer(or only do) 129. Many have
done this including myself. I started with the C and get pretty much
everything done and then found out
through sequencing that the sequence was virtually identical to B6. But the
problem was (likely happen to
many genes), my long arm was in promoter region, containing numerous AT or
GC rich region or repeats, so it
was very hard to PCR all of them out in long pieces(BTW, for reference
【在 g***y 的大作中提到】
: 多年没做这个了，知识比较老化。
: 1.比较两个arm的sequence homology between 129 and B6.
: 一般的担忧是因为两个strain的序列有差别，可能影响同源重组的效率。但是如果幸运
: 的话也许你感兴趣的区域两者高度同源。
: 否则，几个OPTIONs
: A。选用B6 derived ES CELL,有些service提供的。
: B.让你的arm尽量长. 但是还是不能保证一定会有positive clone,要看运气，并且问题
: 是plasmid大到一定程度就不稳定了。比较safe的办法是用BAC recombineering做
: BAC based construct.
: C。 use long-range high fidelity DNA polymerase to PCR 129 genomic sequence.
| r**h
发帖数: 6 | 5 Thanks. Good to know all about your experiences and thoughts.
I guess that the sequences with apparently essential roles could be quite
conserved between 129 and B6, say, protein coding sequences, and perhaps the
promoter region you mentioned (certainly I need to carefully check with
this claim). This could be one of the reasons why you were able to go with
B6, a fortunate move. So, maybe I should take my chance to grab a B6 clone,
because the region I am eyeing for targeting is pretty close to
【在 n********k 的大作中提到】
: All good suggestions...I would vote for the second one as many core facility
: prefer(or only do) 129. Many have
: done this including myself. I started with the C and get pretty much
: everything done and then found out
: through sequencing that the sequence was virtually identical to B6. But the
: problem was (likely happen to
: many genes), my long arm was in promoter region, containing numerous AT or
: GC rich region or repeats, so it
: was very hard to PCR all of them out in long pieces(BTW, for reference
| n********k
发帖数: 2818 | 6 First, to clarify a bit, I didn't (or meant to) claim the promoter region
very likely conserved( as it turns out,
many are but evolution has also made use of promoter changes to do tricks
as people are starting to
appreciate). Secondly, (from some of your description), are u targeting
for non-coding RNA, or splicing site
or promoter/enhancer region like that? Then that could be quite different
and I would take back much of
what I had said until you know ur targeted region is absolutely conse
【在 r**h 的大作中提到】
: Thanks. Good to know all about your experiences and thoughts.
: I guess that the sequences with apparently essential roles could be quite
: conserved between 129 and B6, say, protein coding sequences, and perhaps the
: promoter region you mentioned (certainly I need to carefully check with
: this claim). This could be one of the reasons why you were able to go with
: B6, a fortunate move. So, maybe I should take my chance to grab a B6 clone,
: because the region I am eyeing for targeting is pretty close to
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