z*******g
发帖数: 224 | 1 【 以下文字转载自 Love 讨论区 】
【 原文由 cDNA 所发表 】
就这样结束了。。。
一周两次的通宵电话；
5000多刀的电话费；
寒假特意回国去看她；
以及那个漂亮的结婚钻戒；
都没能阻止这一天的到来。。。
就象她说的，这也许是天意吧。。。。
从来没有象今天这样难受，
虽然我已经历过十几次的分手。
以前总是感到如释重担，
但这次。。。。。。。
上天是公平的，
他让我一次还清了所有欠下的债。
静静地收起她的照片，
笑声，泪水，一切美好的回忆都会随风而去。
在清华的最后半年是我生命中最美好的日子，
可是我没有好好珍惜。
只有当失去她的时候，才感到她的可贵。
她送我的玉佩，还挂在我的项间，
我曾答应过她，不娶到她，决不摘下。
所以我会永远带着它，
提醒我，
人世间，情为何物。。。。 |
|
w***g
发帖数: 712 | 2 武汉大学植物发育生物学特聘教授: 吕应堂
简 历 吕应堂，男,武汉大学生命科学学院教授、博士生导师。1957年9月出生于湖北省新洲县。1981年在武汉大学获理学学士学位,1984年在中国科学院北京遗传研究所获硕士学位,1991年在美国夏威夷大学植物分子生理系获博士学位,1991年至1996年在美国加州大
学伯克利分校植物生物学系做博士后研究员。1997年回国,获国家杰出青年科学基金资助。现任武汉大学发育生物学研究中心主任。
主要成就 1.植物钙/钙调素介导信号传导组成因子的鉴定及其生物学功能分析首次获得编码植物钙调素结合蛋白的cDNAs,为探索植物钙/钙调素介导的信号传导途径的作用机理奠定了分子基础,并确定了植物钙调素结合蛋白的钙调素结合功能区，证明了钙/钙调素系统在植物热激反应中的调控作用。
2.植物生长发育过程中光信号传导分子基础的探索利用光依赖性玉米根向地性生长为研究对象,通过生理学、生物化学和分子生物学分析,表明钙调素依赖型蛋白激酶在光信号传导中的可能作用。
3.植物开花控制的分子生物学研究成功地分离出玉米、烟草和水稻的钙/钙调素蛋白激酶基因,揭示钙/钙调素蛋白激 |
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W***o
发帖数: 6519 | 3 是不是还要参照cDNA的量来normalize一下？
5年多没搞这东西了，呵呵 |
|
W***o
发帖数: 6519 | 4 是不是还要参照cDNA的量来normalize一下？
5年多没搞这东西了，呵呵 |
|
c**A
发帖数: 1474 | 5 【 以下文字转载自 Windows 讨论区 】
发信人: cDNA (照镜见白发), 信区: Windows
标 题: 中文windows7的烦恼
发信站: BBS 未名空间站 (Sun Mar 14 13:23:13 2010, 美东)
turbotax 2009 装不上.
需要英文版的 .NET framwork 3.5 sp1
可是根本没办法在windowns7 上装卸不用语言的 .NET framwork..
郁闷....
不知道有没有人遇到同样的问题,如何解决? |
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n*******e
发帖数: 27 | 6 i have only used RNaseH, RNaseT1 for cDNA stuff. and i donno
its function in mammalian.
let us see this one
Mol Cell Biol 1999 Dec;19(12):8361-71
in S.C., RNase H was found to remove RNA primer from Okazaki frag.
Biochemistry 1999 Nov 16;38(46):15097-103
for HIV and other retrovirus, RNase H function is essential for
activity of the RT.
J Biol Chem 1999 Oct 1;274(40):28270-8
Human RNase H was cloned and found to cleave DNA:RNA substrate,
then what function:)? antibody template switch? DNA replic |
|
y*******n
发帖数: 22 | 7 [问题] 给定N=m*n个相同长度的字符串(例如L=20)，欲找到一种算法
，
把它们排列到一个m*n的方阵上，使得所有相邻两个字符串的距离之
和最小。此处两个字符
串的距离定义为它们之间不同的字符位数，例如
dist(ABCDCGA,ABEDCAA) = 2.
[背景] Affymetrix生产的基因芯片基本原理是针对一个物种的每一
个基因(可以考虑成ACTG
组成的字符串)设计一组特定的引物(primer)(即一系列长度一定的字
符串)，然后在一块固
定大小的载体(如玻璃或尼龙)上并行合成这些引物。实验时把载体同
这种生物细胞中的mRNA
(或RT-PCR后的cDNA，经过染料修饰)杂交，根据每个引物杂交信号的
大小来推断各个基因在
细胞中的表达量。上述问题与Affymetrix的芯片合成技术有关。
考虑在2*2的方阵上合成如下设计好的引物，用1234来表示四个孔的
编号，
AC CC 1 2
AG GC 3 4
通过这样一个方案：
(1)在溶液中放核甘酸A，打开样孔1，3，这样，1，3样孔里就合成上
A：
A -
A -
(2)在溶液中放核甘酸G，打开样孔3，4，这样，3，4样孔 |
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n***h
发帖数: 11 | 8 照我的记忆说来：
yeast 2 hybrid system是两个蛋白之间的binding
yeast 1 hybrid是蛋白质和DNA之间的binding ，就象ras说的，测出的是bait和
fragment之间的结合，可以象ras说的那样fragment是library, 来找和已知蛋白
结合的特定element，也可以特定的fragment，转cDNA library进去，找和这个
fragment 结合的蛋白。 （infact that's what I did)
回来说2hybrid。transcriptional factor一般至少有两个domain: binding domain
和activition domain. binding domain binds to DNA elements, while activation
domain activates the transcription of downstream genes. 我们要测试蛋白A
和蛋白B是否bind, 就做以下两个construct：
—————————— ————————— |
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n***h
发帖数: 11 | 9 照我的记忆说来：
yeast 2 hybrid system是两个蛋白之间的binding
yeast 1 hybrid是蛋白质和DNA之间的binding ，就象ras说的，测出的是bait和
fragment之间的结合，可以象ras说的那样fragment是library, 来找和已知蛋白
结合的特定element，也可以特定的fragment，转cDNA library进去，找和这个
fragment 结合的蛋白。 （infact that's what I did)
回来说2hybrid。transcriptional factor一般至少有两个domain: binding domain
和activition domain. binding domain binds to DNA elements, while activation
domain activates the transcription of downstream genes. 我们要测试蛋白A
和蛋白B是否bind, 就做以下两个construct：
—————————— —————————... 阅读全帖 |
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l*****k
发帖数: 587 | 10 how about do southern to get physical restriction map(if you have probe for the
gene you want?)
Actually the question you asked is weird, to me :)
if you want to do funsion construct for expression, should you use cDNA??? why
you bother to go after BAC, I think they are all genomic DNA
for expression, the best system I ever used is the PET30(a,b,c) system, all my
proteins got very strong expression, HIS6 helps to purify protein using Ni+
column, thrombin can be used to remove the fursion part fr |
|
g***m
发帖数: 465 | 11 hehe, it is a basic strategy via accurate MS for proteomic level's screening
and identifying.
The precision is really dependant on how you construct the peptide map
database. Generally, they take cDNA translated amino acid sequence as actual
proteins, then do a in silica proteolysis with trypsin, input the results into
the database.
This approach is really database dominating. Not mention possible
post-translational modification and signal peptides and so on that could bring
a big trouble. |
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M****e
发帖数: 70 | 12 this is a BIG problem. so what is the gene? and from what
kind of organism would u like to clone it? i have only a
couple of suggestions. first of all, check the RACE primers.
have you tried several different primers? 2nd, check the
known EST database and see whether there are severa image
clones available with which you can actually assemble the
clone. this gene might have very long 5'UTR. you may not
need the whole sequence for expression, though the UTR may
have regulatory sequences. 3rd, you |
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g***m
发帖数: 465 | 13 check NEB catalog for Taq enzyme, it has double strand specific 5'->3'
exonuclease activity.
My guess is primer and/or product degradation by Taq.
BTW, one small tip for pfx is: using 2X buffer rather than 1X. I got several
cDNA clones by PCR, and none of them disrupt the ORF by this modification.
DNA(WASP)
然后用同样的primer
colony-pGEX4t1
这些在处理后的pGEX4t1 |
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c*****t
发帖数: 1084 | 14 for a long time people already know that the presence of an intron will
signicantly increase the expression level of genes. the classic example is
beta-globin. w/o intro its cDNA barely expresses in cells. with an intron
even an irrelevant intron (insulin intron for example) it expresses at very
high level. you may wanna search with key word "intron, expression (beta-globin)
but i don't know much what's the situation in transgenic mice though. |
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g****r
发帖数: 14 | 15 Oh, I omit an E
it is I.M.A.G.E. -you can check the clone out in ncbi, to see if there is information
on this cDNA/entry, if there is something like an image number, or atcc number
then you can find the clone from atcc website, order it, about $50~ bacteria ... |
|
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f***r
发帖数: 19 | 17 1) Do your realtime primer/probe span exons? If not, genome contamination may
cause the discrepancy.
2) Narrow down your standard curve range, for example using a two or three
times dilution intead of ten.
3) It seems you use absolute standard curve method. If you just wanna compare
two samples, why not use total cDNA make standard curve and normalize to
housekeeping gene? |
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n********k
发帖数: 2818 | 18 guess you shall have asked your boss before you do anything, all
communications like this generally should be initiated by your boss (until you
are the boss, are u::). By the way, my experiences with Japanese and others as
well like this are quite positive actually. If you don't want to tell them
what you want to do, then be smart about it rather lie: be general and avoid
specifics. to lie is not a good way to go for sure i guess unless you never
publish and it is not professional either by the |
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p*****n
发帖数: 981 | 19 ☆─────────────────────────────────────☆
smilingdog (smilingdog) 于 (Sat Jan 20 22:41:27 2007) 提到:
在做一个未知功能的基因，好像不属于任何已知的family,sequenc和其他的cDNA/蛋白
相似度也很低。不是到有没有什么办法能预测这个基因的功能？比如可以找到什么
microdomain吗？或者这个基因的某一部分和别的蛋白相似？有什么生物信息学的办法？
已经做了两年了，毫无头绪啊。。。
☆─────────────────────────────────────☆
Acartia (深水鱼) 于 (Sat Jan 20 22:43:17 2007) 提到:
要是pseudogene呢？
法？
☆─────────────────────────────────────☆
raison () 于 (Sat Jan 20 22:49:15 2007) 提到:
有一些预测基因功能的软件，基本上都是crap, 恐怕只能通过interacting protein 的 |
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t****p
发帖数: 1504 | 20 导读：有很多生物学工作者尽管知道高通量技术的大概原理，但是却以为它仅仅是更快
而已。其实高通量DNA测序技术不仅仅是替换传统的测序技术，它有三大优点是毛细管
测序法所不具备的。首先它把平行处理的思想用到极致，可以在几百万个点上同时阅读
测序，所以它是快速高通量的技术，这一点是大家所熟知的；另外它实质上是单分子
DNA的测序技术，通过单分子DNA结合在某个点上进行扩增测序，所以它可以测一个DNA
的mixture（混合）；最后由于在一个 mixture中某种DNA的丰度反应在它能在多少点上
结合并被测序，也就是说被测序的次数反应了DNA的丰度，这个技术还有非常完美的定
量功能。
高通量DNA测序技术已经在如下领域掀起了革命。
测基因组的：
1. re-annotation of the genome。测序难免有错误，通过反复的深度测序，可以将这
种错误降低到很小。另外通过反复测cDNA library，纠正了以前splicing位点的错误。
大规模地，便宜地，快速地测物种的序列，比如果蝇的subspecies的序列，对于理解进
化非常有必要。
2. 疾病相关基因以及突变的hot spot |
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t*x
发帖数: 1065 | 21 Thanks a lot!!! Really appreciate!
You are totally right....The relative Ct seems remain same and the dye
actually is added together with the premix. The reason why I worry this so
much is that the Ct value of my target gene is so late (>35) even though I'
ve already increase the concentration of cDNA so much. |
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t*x
发帖数: 1065 | 22 Thanks. I changed but results remain similar.
Is it okay that I load 2-4ug cDNA/reaction? I am afraid it is too much and
is so abnormal. |
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b****x
发帖数: 110 | 23 I think it is not necessary to load 2-4ug cDNA. It is too much even if you
get a relatively normal Ct value.
Checking the machine settings for all parameters is helpful. |
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t*x
发帖数: 1065 | 24 Thanks for your response.
Yeah, I also concern about that. But if I load less cDNA, there is no
amplification curve before 40 cycles at all.
The machine settings did not change at all, but I'll double check. Thanks! |
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A******y
发帖数: 2041 | 25 If you are using one-step, Quanta Biosciences kits are the best. I have
used all major brands (new Bio-rad, Applied Bio, Invitrogen, etc). Quanta
is the OEM for Bio-rad for long time. You also want to make sure that you
have two housekeeping genes and normalize your mRNA (cDNA) loads as a third
control. I like GAPDH and S18 rRNA. |
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n**********1
发帖数: 251 | 26 实验室要进行microarray，标本要求合成到双链DNA，第一链DNA合成应该就是普通
的cDNA合成吧？ 但是不知道第二链是否有现成的试剂盒，有经验的朋友推荐一下。多
谢了啊。 |
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n**********1
发帖数: 251 | 27 做过的人提示一下啊，就是大家用什么试剂盒来合成用于microarray的双链cDNA啊
，多谢了啊。 |
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l******u
发帖数: 936 | 28 不难，好像是有些公司的机器可以直接测RNA了， 有些公司的机器要把RNA搞成cDNA才
能测。 |
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d*******w
发帖数: 45 | 29 想分析一蛋白在人肿瘤中的表达情况，已经做了immunohistochemistry，还想分析一下
肿瘤中mRNA的水平. 有人买过吗？谢谢！ |
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A***g
发帖数: 191 | 30 做了二十多个基因在十几个不同的人的细胞系中的表达量实验。
用的是qPCR方法。我的做法是，以cDNA为模板，以GAPDH为internal control，做看家
基因的模板多稀释20倍。
结果分析呢，我用的是 2-△△CT Method，设定某个基因在某个细胞系中的表达量为1
（这个细胞系也就是我设定的对照），那该基因在不同细胞系中的表达量就都是相对这
个细胞系而言，这样得出来的结果是relative expression level.
可是老板要的结果是最好能一目了然能看清不同基因在同一细胞系中的表达量变化，同
时也能兼顾在其它细胞系中的表达量变化。她说我给她的结果很容易让人误解。因为每
个基因都有一个细胞系为1，看上去好像这两个基因表达量一样似的。
我用的这个公式应该不能够比较不同基因的表达量吧？
同时我为了结果好看，基本上都是选择表达量比较低的细胞系为1，这样有的表达量高
的数值就很大，我老板就以为这个数字这么打是不是表达量很高很高啊？
我家老板要达到的这个目的要怎么分析呢？有没有相关文献啊？ |
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e********r
发帖数: 147 | 31 不好意思，这个RT-PCR实验有点粗，是分别用sense和antisense引物去做cDNA第一链的
合成，然后再做PCR。这样可以分别检测antisense和sense的RNA产物。应该用Northern
进一步验证的。
这个antisense能把翻译统统都block掉，也太牛了....
我是不是应该试试其它的培养条件，或者想办法阻止antisense的转录什么的？ |
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h****r
发帖数: 152 | 32 用pcr引物去做RT是挺粗的，PCR的引物一般都很长，而RT的反应温度比较低，如果你的
sense引物的3'端GC含量稍微高点就会有非特异性的配对。不信你可以用其他基因的引
物在你的sense引物合成的cDNA第一链中扩增试试，也许也可以扩增出来。
Northern |
|
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c***y
发帖数: 615 | 34 I mean I am trying to determine primer efficiency using the same batch of
cDNA at different days. They could be vary from 50% to 90%. Based on Qiagen
qPCR protocol, all primer efficiency should be above 83% (the corresponding
slope is less than 3.8).
Every time I did serial dilution, R^2 is pretty good; but slope number
differs markedly time by time.
robust
cycles |
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A******y
发帖数: 2041 | 35 First, when you do your titration, do you know your absolute cDNA
concentration? or every time you use different concentration? What kind of
water are you using? Are you using a filtered tip? What's your cycle like
and how's the melt?
I don't think you have to prep the PCR on a cold plate since you are doing
one step but maybe is ideal to keep it a 4 degree. |
|
c***y
发帖数: 615 | 36 DNA concentration is determined by Nanodrop. And I compared the efficiency
from same batch of cDNA.
I used DNase/RNase free water order from some company (don't remember
exactly since I am not at lab), and filtered tip.
The cycle number is between 20 to 25, depends on the primer set. The melt
curves looked fine.
Why do you think 4 degree prep is ideal? Thanks
of
like |
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p*****m
发帖数: 7030 | 37 1 你google一下从EPO receptor到serotonin receptor到TRP channel怎么搞出来的
就行了 简单来说就是搞一个没有receptor的cell line，把有receptor表达的cDNA
library转进去 然后加ligand，看下游的phenotype就行了 可以是binding activ
ity，也可以是downstream signaling,等等
2 没有 但是如果你有个范围可以大概用agonist/antigonist猜猜看
specific |
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p****l
发帖数: 291 | 38 yeah, it is really difficult. One of my projects is quite similar.
I finished the step crosslink and IP, and sent the sample for MASS.
But to confirm the receptor is the protein reported by MASS, still need a
lot of time.
I prefer the method you recommended, transfer cDNA library to a cell line
which does not express the receptor. Screen the positive clone. |
|
h**********r
发帖数: 671 | 39 问大侠一个问题，我想从拟楠芥中克隆多个基因分别在微生物中表达。请问有没有省事
和比较快的方法得到这些cDNA呢？有没有这样的文库呢？
多谢！ |
|
v***a
发帖数: 1242 | 40 在NCBI上查到蛋白的mRNA序列，要设计引物以便引入Expression Vector中。
但这个蛋白我不需要全长，而是仅需要它的一部分，还得在比较精确的位置停止PCR的
反应，所以我想在要它停的地方引入一个TGA的stop codon。我平时设计anti-sense的
引物都是根据在NCBI上查到的mRNA序列从3'开始reverse读出一段作为引物，要引入此
stop codo的话，我有点想不通该怎么引入了，是要把TGA反向变为ACT加在平时正常的
引物后吗？
还有一个白痴问题，为什么我查到的mRNA序列会和我在别人paper上看到的cDNA序列一
模一样？
谢谢大家。 |
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v***a
发帖数: 1242 | 41 谢谢。
mRNA和cDNA不应当是互补的么？
你的意思是直接把TGA加在设计好的引物3'末端就可以了是吗？ |
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a****d
发帖数: 1919 | 42 多谢建议！
我试试在ATG之前找另一段primer
cDNA |
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T*G
发帖数: 600 | 43 make a synthetic one if the purpose is to express a protein, keep AA same
while replace compatible
nucleotide from GC to AT
cDNA |
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a********k
发帖数: 2273 | 44 用Herculase II的cDNA扩增的条件，或者clontech的advantage 2
我忘记我克隆GC rich用的哪个了，后者PCR产物肯定非常多，不过突变率有点高
带。 |
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n********k
发帖数: 2818 | 45 1. synthesis or any cDNA clones outthere...or just synthesis the little
piece and then fusion PCR to the entire piece...
2. Primers ahead of the region
3. Primers after this region but with this long tails and go with two
step PCR: 94 68, also try Primestar,
4. PCR small fragments and then fusion PCR...
5. PCR other regions and then add this little piece through PCR-
mutagenesis...
6. Get a bac/genome clone for this gene and PCR in small pieces and
then Fusion PCR, or just PCR this hard region a |
|
C*******e
发帖数: 4348 | 46 就提个醒哈
也许lz已经考虑过这个问题了
cDNA文库好不好？是不是自己准备的（或者可以信任的人准备的），做模板的时候有没
有加太多，加多了可能抑制PCR反应 |
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f***n
发帖数: 34 | 47 Yes. You maybe need to redesing primers. Also you need know that 5' RACE is
more challenging. Only for 3' RACE, i bought all the reagents from different
companies and combine to do it and it is cheaper. For 5' RACE, it is very
important to get full length mRNA or cDNA. Some kits can help you to achieve
this. |
|
f***n
发帖数: 34 | 48 I don't like clontech's RACE kit. Based on my expericne, a lot of cDNA are
not full length. |
|
f***n
发帖数: 34 | 49 If your expression level is high, you don't have any problem with Clontech's
, but if your expressin level is very low, you will get proble. Also if you
use Clontech's kit to produce cDNA library, you will find that a lot of
clones are not full length, however abion's avoid this problem. This is just
from my experience. |
|
t******r
发帖数: 8600 | 50 Fosmid & BAC libray都做了三个了。能筛出其他基因，但从来没找到这clone的影子。
没有
genomic probe, 只有cDNA probe.
PCR是分段进行的。 |
|
