s**c
发帖数: 34339 | 1 ☆─────────────────────────────────────☆
sioc (煎饼爱果子) 于 (Wed Jun 20 13:05:00 2012, 美东) 提到:
上半区：
捷克 6.21 2:45 葡萄牙 -----
半决赛 ------
西班牙 6.23 2:45 法国 ----- |
|
下半区： ----- 决赛
|
德国 6.22 2:45 希腊 ----- |
半决赛 ------
英格兰 6.24 2:45 意大利 -----
规则... 阅读全帖 |
|
j**d
发帖数: 825 | 2 我们公司需要做oligo的人，现在就要人，不过你的本事太大，这里庙小，呵呵 |
|
m******5
发帖数: 1383 | 3 两个月后赴美读PhD，在犹豫是在国内买别的牌子的笔记本还是抵达美国后买mac
主要还是常用的一些软件是否好解决的问题（是否有好下载到的盗版）
sibelius 6 以及常用音色包
常用生物软件全套，比如laser gene ,oligo pathway studio |
|
g***m
发帖数: 465 | 4 the principle is quite the same as DNA microarray except it
employs protein-protein interation in stead of annealing of
oligo DNA. Normally a chip coated with epitopes is used to
detect a cell flow. The opitic character will change if any
cell surfact would interact with any epitope on the chip. It
is very cool to study the surface protein interaction. Johns
Hopkins has a website and service for this tech.
It is a nascent technique as I know.
Another powerful tech for protein identification is m |
|
d****o
发帖数: 361 | 5 From which company did you order your primer?
we order from Invitrogen, and they use different salt concentration
to calculate Tm. It's not about right or wrong, coz Tm changes with
oligonucleotide and salt concentrations, so u'd better calculate it by
yourself. Some softwares like Vector NTI can do it, I think Primer3
works too.
we use oligo conc. at 250 pM and salt conc. at 250 pM to calculate
the Tms. |
|
M****e
发帖数: 70 | 6 you are right. the difference is exclusion size. protein or
molecule that has bigger size than 25kD globin protein cannot
enter G25, and thus pass through the column without delay. the
oligos are pretty small and can be held in both G25 and G50
columns. so why do they recommend G25 instead of G50? |
|
m**o
发帖数: 13 | 7 Whether or not the probe is excessive basically depends on the level of the
targetting DNA. Generally I believe the concentration of your probe should be
enough for the detecting because 10ng/ml of a 16mer oligo aproximately equals
to 1.5nM by molar concentration, which could capture 1.5pmol targetting DNA if
it bound at a ratio of 1 to 1.
To label or not to label, it depends on the method you used to detect the
targetting. If you use, for example, biotin to lable to probe, then label the
probe |
|
h******b
发帖数: 312 | 8 There are primarily 2 types of microarray, comercial Affymetrix chip and
spotted array(cDNA/pcr), I guess u mean spotted array.
two major differences between the 2 types:
1. spotted material: affy use de novo synthesized oligo, 20mer or 25 mer,
called a probe, each gene is represented by 11? or 16 probes. the whole thing
called a probeset, so a probeset correspond to a gene. spotted array is
relative flexible, can print on the chip cDNA(comercially synthesized) or pcr
product, or even promoter s |
|
T****u
发帖数: 424 | 9 1。size和Y 一样大吗？
2。有没有Y-N terminal的抗体？have a try
3。knockdown using different oligos targeting different regions of Y, using
antibody to detect. |
|
s******y
发帖数: 28562 | 10 Thanks! I will give them a try later.
Are they using the oligo-dT system or the 5'-end RNA ligase?
Is it possible to do a 5-RACE with Fermentas' kit? |
|
h******u
发帖数: 602 | 11 The one we currently have is from Promega nad the concentration is: 20–100u
/μl.
I plan to use 1 unit, 10 unit and 100 unit per reaction (0.5ug oligo) and
incubate on ice, room temperature and 37 C. Is this OK? |
|
O******e
发帖数: 4845 | 12 看看别人的PAPER，看有没有已经被人用过的顺讯，然后自几找个公司合成就是了。
如果非要买别人预制的，哪家都差不多了。他们会给你MANUAL，照着做就是了。
5nmol/each oligo is more than enough for you to try their efficiency. |
|
b******k
发帖数: 1874 | 13 好像直接合成oligo，然后anneal，再链接就行。不过我也没有具体干过。我看的文章也
没有具体说怎么样anneal的。。。。 |
|
a***e
发帖数: 1010 | 14 when u order oligo, the end is -OH, you have to kinase first by T4 PNK.
you can kinase (+) & (-) strands together in 50 ul volumn. then boil it
in water, let the water cool down on bench gradulately.
then it is enought for ligation. |
|
b******k
发帖数: 1874 | 15 so, if i am going to ligate two tandem short fragments (say, 50bp each):
synthesize 50nt oligo of Sense and antisense (with 5' phosphate group modifi
cation),
anneal them by boiling and cooling naturally,
then ligate the annealed fragments (ideally, with synthesizd 3' overhang fro
m first one, and 5' overhang from 2nd)by conventional T4 ligase system?
thanks. |
|
b******k
发帖数: 1874 | 16 i will post it again when back home. sorry, sounds my pc in schoool doesnt'
support chinese font.
s
t
r |
|
s******y
发帖数: 28562 | 17 Sorry I cannot give out any clear reference for plant protein being
difficult to denatured.
The thing is plant cells have cell walls, that's very different from
animal cells. Many proteins in plant cells are also N-oligo-glycosylated,
which is quite different from animal cell.
That idea was based on what people told me when I used to work in
a plant biology lab long time ago and I assumed that is a popular
assumption in the plant biology field. Maybe someone in this board
who is currently workin |
|
i*****l
发帖数: 51 | 18 艳阳天MM的回答准确，就按她的建议去做。 T4 PNK是用来末端磷酸化的，公司合成的
REGULAR OLIGO都是-OH末端， 直接和载体连接就可了
kinase
了呢？ |
|
s******y
发帖数: 28562 | 19 DNA are double helix
On the vector part there will be one 5'-end and one 3'-end on each side.
If only one end has the phosphate group, it will be enough for the ligation
to work. The nicked end can be patched up later inside the bacteria.
In some case, in order to increase the efficiency of transformation, people
use T4 ligase to put the phosphate group on the two 5'-ends of the oligo. I
think this is why your senior in the lab ask you to do that. In this case,
you should follow his/her directio |
|
c*****a
发帖数: 179 | 20 想把一个target基因和若干oligo在454 sequencing的结果里面进行比对，请问要怎么
实现？
看了会儿mega的help还是不得要领
或者谁有其他的好软件推荐。谢谢啦 |
|
q*****d
发帖数: 445 | 21 请教大家一个技术问题。我用过一个荧光定量PCR试剂盒，效果挺不错，想了解一下引
物和探针的具体序
列，但是试剂盒里这些oligo DNA是混一起的，质谱很难分开。想了一个办法，把扩增
产物上T载测
序，就知道引物的位置，但是具体序列上只知道5‘端开始的序列，却不知引物的3‘端
序列具体在哪里终
止，因为一般引物大约18-25 nt长度，有很多的可能性。各位有没有什么办法解决这个
问题？ |
|
s******y
发帖数: 28562 | 22 There is no easy way.
In theory you can seperate the oligos with HPLC, then mass spec them one by
one |
|
a****o
发帖数: 1786 | 23 prepare one tube of 100uM and one tube of 10uM. when you run out of 10uM
working primer, make new one from the 100uM stock. DNA oligo is very tough.
don't worry about it. |
|
c****1
发帖数: 1095 | 24 貌似IDT口碑不错。你们都在他家合成oligo么？我们刚从另外一家公司转到IDT了。 |
|
x*****e
发帖数: 309 | 25 很多时候引物设计很重要，与其优化半天不如重新仔细设计引物，oligo 软件不错, 我认识个师兄一直停留在复制粘帖阶段，说引物不需要设计，无语。 |
|
O******e
发帖数: 4845 | 26 Off-target effects might have contributed to what you got. I would try
several
more siRNA oligos targeting different regions of your gene to confirm the
death phenotype. At the same time, try to rescue with resistant constructs.
siRNA |
|
T**********t
发帖数: 1604 | 27 看RNA degradation好像一般都是用agilent的bioanalyzer比较准，用量很少，结果快
而且直观，就是他家的chip和仪器比较finicky，操作起来要很小心，否则一点小纰漏
就废掉一块chip。
如果是细胞里提的总RNA，bioanalyzer的分析软件能给出RIN数据，叫RNA integrity
number，数字越低，RNA降解得越厉害。
但我估计你看的RNA是short RNA oligo library，可能不会有RIN吧，我没试过。但是
你的sample如果用analyzer跑一下，出来的结果可以像胶一样看，有没有degradation
应该很容易看到吧。只需要几分钟的时间，有条件的话，试试看吧。 |
|
d**********t
发帖数: 20415 | 28 最近realtime PCR遇到怪事了，请大家帮忙分析下原因：
问题：几个基因的值出现很多undetermined的结果，但是之前一直在同一个cell line
里做，一直都可以检测到
条件：Realtime的template是RNA反转录的cDNA
cell line和RNA抽提的方法都和以前一样，处理方法也一致
引物用的是刚刚从stock里面稀释出来的，和之前的实验来自同一管stock
拿之前证明可以检测到的RNA作为阳性对照，也是undetermined
反转录kit整个实验室公用，其他人没有遇到问题
反转录试过oligo dT和random primer，结果没有差别
其中一个基因刚刚设计了一对新引物结果也是undetermined
作为positive control的基因可以检测到没有问题
多谢了
如果需要更多细节，我会补充 |
|
s***m
发帖数: 6197 | 29 问题解决了没？
你说公司买的vector是什么意思？你给他们序列，他们给你合成的oligo？IDTDNA订的？
你第一次做出来的还保留着没？如果有可以试着gel purified一下，然后用它来做模板 |
|
y******8
发帖数: 1764 | 30 Have you tried other methods?
Single biotinylation could be insufficient. |
|
w******e
发帖数: 1187 | 31 by other methods you mean other than biotin-avidin? I chose this
as I thought it's the easiest...
I didn't know that... do you know the reason ? thx! |
|
w******e
发帖数: 1187 | 32 thx a lot! I also assume the interaction to be robust, so I didn't
use the recommended buffer condition.... How long do you let the incubation
go? did you do NP conjugation or slide? How did you measure conjugation
efficiency? I had to use qRT-PCR everytime:(
sorry for more qs:P |
|
y******8
发帖数: 1764 | 33 不知道你要多高的效率？双生物素偶联效果也许会好很多。
另外，提高反应温度也许会有帮助。
没想过用amine group吗？ |
|
w******e
发帖数: 1187 | 34 起码希望有一小半能conjugation上吧。amine group也考虑过，不过是不是
只能在synthesis时加上？post synthesis往RNA上加amine容易吗？
谢谢！ |
|
w******e
发帖数: 1187 | 35 是这样的：我的RNA是2'-F modified aptamer，80base左右，据说找公司合成
很贵（不过也没问过）。我现在加biotin是用kit加在3’end。即使找人合成
也只敢往5’/3’上加，加到中间怕破坏结构：（
PCR |
|
T**********t
发帖数: 1604 | 36 我做30min的incubation.
我用的是dynal beads，应该没有到nano的级别吧，估计微米级？
binding efficiency我就是测一下binding前后的UV差值。我做得没那么精细，就是毛
估估差不多就好了，反正是library，多一点少一点看不大出来。。。
incubation |
|
y******8
发帖数: 1764 | 37 我的直觉是加biotin反应不完全。
建议直接合成。
猜想，可以用一个反义短DNA链做模板，用polymerase加多个biotin-T在3’端。
如果成功了，记得给个包子。 |
|
w******e
发帖数: 1187 | 38 thanks for the suggestion. I'm been struggling whether should conjugate
my primer for the aptamer to the beads and then use base pairing, as
in theory that may affect structure too:( |
|
Z******5
发帖数: 435 | 39 前几天刚用invitrogen的5´ RACE System for Rapid Amplification of cDNA
Ends, Version 2.0搞定一个13kb的RACE出来。
RNA质量很重要；要是只做反转录的话，invitrogen的SuperScriptTM Ⅲ First-Strand
Synthesis System可能更好；可以考虑在3'UTR位置多设计几条Gene Specific
Primers，效果比Oligo dT应该要好很多；多设计几条nested PCR引物，进行多轮扩增。 |
|
p********i
发帖数: 116 | 40 第一次用合成得oligo做RNAi，使用lipofectamine 2k转的293，转染的量按照说明书来
的，但是没有效果。看
到很多paper上面都是转多少nM，我是转了100pMol，会不是太少了，还有paper中的nM
这个写法应该是终浓
度吧，到底应该转多少合适呢？看到dharmacon上面推荐终浓度是25nM。谢谢。 |
|
O******e
发帖数: 4845 | 41 简单的说，一个是增加某基因的表达，一个是减少某基因的表达，但达到这两个目的的
方式－－delivery－－几乎是一样的。你如果只是简单地送几个siRNA oligos过去，
效果很快就没有了；可你如果想用其它方式比如病毒载体，那一样是leading to
cancers了。 |
|
b**y
发帖数: 86 | 42 最近刚开始做EMSA，我用的是Pierce的Lightshift kit. 多次实验都只看到
nonspecifec binding（不能被unlabeled oligo竞争掉）。我发现一个原因可能是
binding reaction里盐浓度过高，因为kit提供的binding buffer（10*）含有500mM
KCl,而我的nuclear extract最后是由high salt buffer提取的，F.C.225mM NaCl。于
是我省略了binding buffer，把最后的NaCl浓度调整在50-75mM，得到了一条specific
的binding，但是非常弱。
问题1. 我看到的paper和protocol都使有类似的binding buffer，而且也用类似方法提
取核蛋白，好像并不考虑nuclear extract里的盐浓度，事实上这个浓度很高。但是，
我的几个sample extract蛋白浓度不同，加入同样多的蛋白的话最终盐浓度就会不同。
大家怎么解决这个问题？透析又怕蛋白容易降解。
问题2. 我加入的nuclear extract的体积无法忽略，大概有10u... 阅读全帖 |
|
s******y
发帖数: 28562 | 43 最好的方法是在那个地方的附近找两个靠近的restriction site,
然后直接合成两段oligo, 中间含有你要的序列，两端粘合后正好形成两个restriction
site
如果没有办法这样做的话，就用3-step ovelapping PCR.要插个20bp还是很容易的，
HF |
|
|
|
|
|
w******e
发帖数: 1187 | 48 想试试用aminoallyl-GMP在synthesize RNA的同时在5'加amine group，方便
下面做各种conjugation(with NHS ester of fluorophore/biotin)，不知有
什么注意事项没有？amine group应该很active 吧？用urea PAGE这种harsh的
条件purify会不会有问题？
多谢！ |
|
w******e
发帖数: 1187 | 49 thx a lot~我买的这个东就是找glen research推荐的，现在正在骚扰生产厂商呵呵。
没什么基础做交叉的题目还挺郁闷的，我们lab虽说号称材料工程化学生物专业的人
都有，可我想做的东西通通没一个人有经验，真ft：（ |
|
