c****d
发帖数: 501 | 1 我们想从一个B cell line克隆IgG1的heavy chain
不知道可不可行
或者如果随便找个donor 用PBMC的RNA呢
本人之前不是做免疫的 还请大家指点 |
m*b
发帖数: 1421 | 2 you can use some commercial kit which provides common primers for
immunoglobulin genes. |
p****l
发帖数: 291 | 3 If you only want to clone IgG1 heavy chain, I think with one step more to
purify B cells from PBMCs will be better than PBMCs.
As for human B cell line, I think most of them should be from B cell
lymphoma. First, you do not know whether they secrete IgG1, second you do
not know how their DNA changed.
I do not know to what extend the gene of IgG1 heavy chain preserved in
different B cell clone, but definitely this is what you must take into
consideration.
And I think it will be better if you can
【在 c****d 的大作中提到】
: 我们想从一个B cell line克隆IgG1的heavy chain
: 不知道可不可行
: 或者如果随便找个donor 用PBMC的RNA呢
: 本人之前不是做免疫的 还请大家指点
|
f**********r
发帖数: 95 | 4 楼主是想克隆重排前的还是重排后的？如果是重排后的一定要保证拿到单克隆才可以。 |
c****d
发帖数: 501 | 5 恩 我们先是自己design了一个长的primer
就是根据pubmed找到的IGHG1的序列
结果还真从我老板的PBMC当中捞到一个clone
就是mutation太多了
不知道你用过这类的primer kit吗
是clone用的 还是一般的genotyping用的 或者是realtime pcr用的呢
我也是糊里糊涂的 但是感觉这些应该是不一样的
【在 m*b 的大作中提到】
: you can use some commercial kit which provides common primers for
: immunoglobulin genes.
|
c****d
发帖数: 501 | 6 first, thanks a lot for the reply!
像我上边说的 我们最初就是用我老板的PBMC提RNA试了一下
用的是clontech的RACE的kit
结果批出来一大堆乱七八糟的东西里 还真捞出来一个IGHG1
可惜和genebank的序列align的结果有很多mutation，大概对上97%
我也不知道是pcr的fidelity的问题 还是个体差异
然后我们就用了CESS cell line, suppose是分泌IgG1的 而且刚好有我们要的membrane
bound form
repeat了同样的试验 这次批出一个非常specific的band， size也对
结果测序之后 竟然是完全不同的东西
blast了一下 发现target到genome上一个未知的loci
然后我就卡到这了 ：（
is
【在 p****l 的大作中提到】
: If you only want to clone IgG1 heavy chain, I think with one step more to
: purify B cells from PBMCs will be better than PBMCs.
: As for human B cell line, I think most of them should be from B cell
: lymphoma. First, you do not know whether they secrete IgG1, second you do
: not know how their DNA changed.
: I do not know to what extend the gene of IgG1 heavy chain preserved in
: different B cell clone, but definitely this is what you must take into
: consideration.
: And I think it will be better if you can
|
c*****i
发帖数: 1392 | 7 try single cell PCR, use commercial IgG primer set |
