a******0
发帖数: 262 | 1 that's exactly the point of reaching out, for no one can have an idea that
he/she knows to be great for certain. besides, many great projects (e.g.
groupon) end up being the idea quite different from the original.
most incubating organizations and angle investors (e.g. y-incubator)
typically value the strength of the team over its original idea, for exactly
the same reason.
sharing is the easiest way to succeed and not wasting your own time working
toward a dead end. the only trick is to find th... 阅读全帖 |
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o**********e
发帖数: 18403 | 2 【 以下文字转载自 CivilSociety 讨论区 】
发信人: onetiemyshoe (onetiemyshoe), 信区: CivilSociety
标 题: 关于最近IBM或出售其半导体业务消息的一些观察 (转载)
发信站: BBS 未名空间站 (Sat Feb 8 12:33:29 2014, 美东)
go88的强文。
总结： 窃行业者诸侯。
I)
发信人: go88 (旧友重来), 信区: SanFrancisco
标 题: 关于最近IBM或出售其半导体业务消息的一些观察
发信站: BBS 未名空间站 (Sat Feb 8 03:58:28 2014, 美东)
这两天新闻报道里提到IBM或出售其半导体业务，不知道大家有谁注意到了这些报道里
提到的潜在买家中，有一家叫global foundries最有可能中标。大家如果开车路过237
和great america parkway，会注意到那里在建一片新的玻璃幕墙的大楼，差不多快建
好了。其中有一座上面巨大的logo，就是global foundries。他们原来的办公楼在237
上880的地方。实际上，就在几天前... 阅读全帖 |
|
B********e
发帖数: 1199 | 3 南加创业峰会既第五届北美UCAHP创业大赛南加州分赛 （2）大会
12.7.2014 圣地亚哥
SoCal Entrepreneurship Summit & The 5th UCAHP Startup21 Contest Semi-final
is on Dec. 7th, 2014 at University of California San Diego, Price Theater.
The Summit aims at stimulating innovation and inspiration in Southern
California, creating a gala of SoCal start-ups and Chinese capital.
The one-day forum consists of investor panels and entrepreneur panels.
Speakers include a number of leading entrepreneurs and investors from San
Diego, Los Angel... 阅读全帖 |
|
g**8
发帖数: 4951 | 4 在上述提到的近日印度政府欲迅速批准foundries项目的新闻里，其决策机构是：the
Minister for Communication and Information Technology 印度通讯与信息科技部。
这里如下是另一条新闻，里面也涉及了这个部门，还涉及了美国高科技大公司的一些
老印高管：
Jan 31, 2013, Newport Beach, California] More than 100 TiE Charter Members,
Members, Guests, and representatives from industry heard from a senior IT
delegation about their new policies and incentives to encourage Electronics
Systems Design and Manufacturing (ESDM) in India. Charter Members and
special Industry guests met with the delegates pri... 阅读全帖 |
|
s******g
发帖数: 755 | 5 InnovationWorks founder and former Googler Kai-Fu Lee took the stage at TC
Disrupt Beijing today with Sarah Lacy to talk about the startup ecosystem in
China.
InnovationWorks is a Chinese incubator focusing on early stage internet
companies in China – interest in startups is exploding, and InnovationWorks
saw 7,000 resumes in response to open applications for its first batch, “
There’s nothing that matches the Chinese entrepreneurs desire for success,
” he said.
Lee sees the future of Innovation... 阅读全帖 |
|
o**********e
发帖数: 18403 | 6 【 以下文字转载自 CivilSociety 讨论区 】
发信人: onetiemyshoe (onetiemyshoe), 信区: CivilSociety
标 题: 关于最近IBM或出售其半导体业务消息的一些观察 (转载)
发信站: BBS 未名空间站 (Sat Feb 8 12:33:29 2014, 美东)
go88的强文。
总结： 窃行业者诸侯。
I)
发信人: go88 (旧友重来), 信区: SanFrancisco
标 题: 关于最近IBM或出售其半导体业务消息的一些观察
发信站: BBS 未名空间站 (Sat Feb 8 03:58:28 2014, 美东)
这两天新闻报道里提到IBM或出售其半导体业务，不知道大家有谁注意到了这些报道里
提到的潜在买家中，有一家叫global foundries最有可能中标。大家如果开车路过237
和great america parkway，会注意到那里在建一片新的玻璃幕墙的大楼，差不多快建
好了。其中有一座上面巨大的logo，就是global foundries。他们原来的办公楼在237
上880的地方。实际上，就在几天前... 阅读全帖 |
|
o**********e
发帖数: 18403 | 7 【 以下文字转载自 CivilSociety 讨论区 】
发信人: onetiemyshoe (onetiemyshoe), 信区: CivilSociety
标 题: 关于最近IBM或出售其半导体业务消息的一些观察 (转载)
发信站: BBS 未名空间站 (Sat Feb 8 12:33:29 2014, 美东)
go88的强文。
总结： 窃行业者诸侯。
I)
发信人: go88 (旧友重来), 信区: SanFrancisco
标 题: 关于最近IBM或出售其半导体业务消息的一些观察
发信站: BBS 未名空间站 (Sat Feb 8 03:58:28 2014, 美东)
这两天新闻报道里提到IBM或出售其半导体业务，不知道大家有谁注意到了这些报道里
提到的潜在买家中，有一家叫global foundries最有可能中标。大家如果开车路过237
和great america parkway，会注意到那里在建一片新的玻璃幕墙的大楼，差不多快建
好了。其中有一座上面巨大的logo，就是global foundries。他们原来的办公楼在237
上880的地方。实际上，就在几天前... 阅读全帖 |
|
a***k
发帖数: 1038 | 8 I
The way led along upon what had once been the embankment of a railroad. But
no train had run upon it for many years. The forest on either side swelled
up the slopes of the embankment and crested across it in a green wave of
trees and bushes. The trail was as narrow as a man's body, and was no more
than a wild-animal runway.
Occasionally, a piece of rusty iron, showing through the forest-mold,
advertised that the rail and the ties still remained. In one place, a ten-
inch tree, bursting through... 阅读全帖 |
|
d**********u
发帖数: 4124 | 9 ☆─────────────────────────────────────☆
cp (cp) 于 (Thu Nov 19 12:24:43 2009, 美东) 提到:
Registration required at:
http://www.tinyurl.com/pkuaanc-cd2
============================================================================================
PKUAANC Career Development Forum: 与百人会成员等探讨职业转型 - 12/4 Friday
PKUAANC Career Development team proudly presents
Career Development Forum Series #2 – Career Changes (Panel Discussion)
The United States offers more opportunities for people in the work force t... 阅读全帖 |
|
g**8
发帖数: 4951 | 10 在上述提到的近日印度政府欲迅速批准foundries项目的新闻里，其决策机构是：the
Minister for Communication and Information Technology 印度通讯与信息科技部。
这里如下是另一条新闻，里面也涉及了这个部门，还涉及了美国高科技大公司的一些
老印高管：
Jan 31, 2013, Newport Beach, California] More than 100 TiE Charter Members,
Members, Guests, and representatives from industry heard from a senior IT
delegation about their new policies and incentives to encourage Electronics
Systems Design and Manufacturing (ESDM) in India. Charter Members and
special Industry guests met with the delegates pri... 阅读全帖 |
|
|
f******2
发帖数: 2455 | 12 看了一下incubation的goal description，感觉google还是就想开源个壳子就把客户赢
过来，估计不会成功。
首先，在这里把spark批评一把：https://cloud.google.com/dataflow/blog/dataflow
-beam-and-spark-comparison
然后，在这里想把spark一统到自己的programming model下来：https://wiki.apache.
org/incubator/BeamProposal
感觉完全不顾databrick的感受。
而且dataflow的server side根本没有开源计划。这就好像azure说，我开源了azure客
户段
，而且是apache项目，你们不要用aws啦。
这么搞在云计算上没法翻盘。 |
|
|
f***r
发帖数: 204 | 14 还在与PVDF战斗中，再次请教有经验的虾们：
终于看到了条带，但是巨多非特异带，比我要的带都强得多。在网上查到多家western系
统的trouble shooting，里面提到的好几条关于high background的建议让我大开眼界，
又困惑非常，因为矛盾很多：
1. 关于tween的使用：有一家建议blocking之后才用tween，否则tween会stick to the
membrane and cause high noise；但很多常规方法都没有提到这一点。
2. 关于blocking reagents和ab incubation buffer：有说dry milk能reduce
sensitivity，所以如果只用在二抗incubation就能很好降低noise，但也有很多全程用dr
y milk的，也有说dry milk与tween联合使用会提高noise的；有说BSA会造成高背景的，
也有说用BSA可以获得理想信噪比的……
3. 关于ponceau s染色：这次为确证transfer有效，用了ponceau s染色，但发现有资料
说ponceau s就算洗干净也会 |
|
l*****k
发帖数: 587 | 15 let me tell you the best way ( I think ):
preabsorption
which means you first incubate your 2nd antibody with your control for several
hours, then use the pre-incubated antibody on your real sample.
it usually gives great results.
hope this helps. |
|
p*****n
发帖数: 981 | 16 ☆─────────────────────────────────────☆
honestman (snail) 于 (Sat Feb 10 22:26:27 2007) 提到:
大家做cell culture trypsinization的时候是：
1. 先加Trypsin, aspirate, 然后inculate， 然后加medium
还是
2. 加Trypsin, inculate， 然后直接加medium
或者是
3. 不同细胞做法不同
谢谢！
☆─────────────────────────────────────☆
honestman (snail) 于 (Sat Feb 10 22:27:08 2007) 提到:
顺便问下， 一般incubate多久？
☆─────────────────────────────────────☆
sunnyday (艳阳天) 于 (Sat Feb 10 22:31:50 2007) 提到:
先加少量，吸出来，再加适当的量，然后 incubate
☆───────────────────────── |
|
s****a
发帖数: 436 | 17 Purification of biotinylated proteins on streptavidin resin: A protocol for
quantitative elution。
For elution of biotinylated proteins, 500 mL
release solution (2% SDS, 30 mM biotin, 50 mM phosphate,
100 mM NaCl, 6 M urea, 2 M thiourea, pH ,12)
were added to the resin and incubated for 15 min at RT,
followed by incubation for 15 min at 96C. Afterwards, the
resin was pelleted by centrifugation for 5 min at 16 100g |
|
w***e
发帖数: 269 | 18 细菌污染是最好辨认的.显微镜下看细菌形状规则,颗粒状,细小,大小均一,就像一层沙
子,要是蛋白质杂质或者细胞碎片,都是大大小小形状无规则.有的细菌长的慢.我有次细
胞感染细菌,每次都是六七天后才很明显.所以你可以把细胞养久一些,确定没有问题之
后再做后面的实验.再贡献一点经验,如果细胞确定感染细菌了,别费心思慢慢排除是什
么原因,也不要省钱.所有的东西(medium, trypsin,serum etc)全部扔掉,都用新的.然
后cell culture hood里面东西全部扔掉,cell culture hood里面仔细用70%酒精擦干一
遍.incubator里面的夹层隔板能拿出来的都拿出来autoclave.再用70%酒精把incubator
里面擦一遍.如果是细菌感染的话,问题应该就被解决了. |
|
c*o
发帖数: 186 | 19 新工作到一个地方，做Hybridoma,时不时有mold出来，也没有大面积的contaminate，
就是一天一，两个well那种。放别人incubator里面的hybridoma没事儿，我自己
incubator里面别的细胞也没事儿。养细胞也不是新手了，以前从来没有遇到过这种情
况。大家帮我分析分析吧。 |
|
C***Y
发帖数: 61 | 20
from
beads
吗？
Here are the details:
Incubate 1at Ab with beads for 1 hr at RT. Wash away the unbound Ab.
Crosslink Ab to beads with Imidoester
Crosslinkers (Pierce). Incubate Nuclear extract (or whole cell lysate) with
beads-Ab.
After washes, elute the co-purified protein complex with low PH glycine
solution instead of boiling beads. |
|
s******y
发帖数: 28562 | 21 Finally! Somebody put a microscope inside the cell incubator and show that
this setting is possible. Continuous imaging of weeks is now possible.
Comopanies are also designing cell incubators coupled to microscope now.
http://www.nature.com/nature/journal/v466/n7310/full/4661137a.html
Cellular imaging: Taking a long, hard look |
|
T**********t
发帖数: 1604 | 22 为什么说finally啊，那篇文章里，最早的尝试在80年代就开始了。
不过我之前倒是不知道在incubator里放个显微镜或者在显微镜下放个incubator原来还
有这么多学问。长知识了。 |
|
r*******w
发帖数: 127 | 23 The same happened to my experiments. The uninfected plates maybe got
contanminated somwhere (in hood or in incubator). When uninfected plates and
infected plates were seperated to culture in different incubators, the
uninfected plates kept normal.
293a |
|
D*a
发帖数: 6830 | 24 自己配的，
网上搜了搜，这是跟我们完全一样的protocol
Final concentration of tail digestion buffer (TDB):
50 mM KCl
10 mM Tris-HCl (pH 9.0)
0.1 % Triton X-100
0.4 mg/ml Proteinase K
Add 100 ul of TDB per tail piece (2-5 mm) in a microcentrifuge tube. Be
careful not to cut too much tail.
Incubate the tube at either 60 C for 3 hours, with gentle mixing every 30
minutes, or 55 C overnight.
Incubate the tube at 94 C (or boil) for 10 minutes to denature the
Proteinase K.
Spin in microcentrifuge at top speed for 15 minutes. |
|
T**********t
发帖数: 1604 | 25 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 |
|
c*****e
发帖数: 436 | 26 it seems the Chemist synthesized this peptide told me that in this peptide
valine or some thing? can be oxidized? (don't know what does this mean).
And I saw from papers other people make similar sample with very complicated
steps with:
"Samples were prepared for the NMR experiments in 6-mm tubes by dissolving 0
.3g of lyophilized material in 1.5ml of distilled H2O. ..The tubes were
placed in a bath at 20C, and argon gas was passed through the samples for 6h
by means of long-tipped pipets placed... 阅读全帖 |
|
l********e
发帖数: 636 | 27 写错了， 应该是10cm, 5ml. the original con of PEI is 10mm. dilute 1000.
prepare 2.4ml DMEM with 0.1ml pei. prepare DNA mixture with 2.5ml DMEM with
DNA. add PEI mixture to DNA mixture dropwise, incubate at RM 20m, then take
the medium out from the plate and add the mixture. after 3-4hour incubate.
take the mixture out and add fresh medium. |
|
w********r
发帖数: 1431 | 28 1.wash cell twice with PBS
2. incubate with DSP/DMSO/PBS solution for 20min at RT (freshly!!! prepared
DSP,2.5mg DSP-->480ul DMSO-->12ml PBS, final 200ug/ml DSP)
3. wash 2XPBS
4.incubate 5min with 50mM Glycine/PBS
5. wash with PBS
6. harvest cell in RIPA buffer with PIM, w/o DTT!!!， followed by CO-IP. Wash stringently with RIPA before WB.
Good luck! |
|
t**a
发帖数: 255 | 29 这个恐怕要具体问题具体分析。Co-IP可变参数很多， 不知道你的问题出在IP上还是抗
体上。要说pre-clean， 可以先把protein和normal mouse/rabbit IgG (depends on
which Ab you will use for IP) incubate for 1-2hr at 4 degree, then add
normal beads and incubate another 1hr at 4 degree, spin and use the
supernatant for next step IP. |
|
n***w
发帖数: 2405 | 30 Thank you guys.
I use PGEX2T vector so GST is on the N-terminus.
I normally do 100ml culture size. Here is how I did this:
1. small culture of the bacteria from glycerol stock O/N, about 6-8ml.
2. transfer the small culture to 100ml 2xYTA medium in the next day morning
, shaking at 300rpm, 37degrees.
3. it ususally takes about 1hr 40min - 2 hrs to have an OD600 around 0.75.
4. add IPTG (stock 100mM) 100ul to get a final concentration of 0.1mM.
5. Lower the temp to 22degrees and shake at 300 rmp... 阅读全帖 |
|
x*****a
发帖数: 972 | 31 最近被一个ELISA kit给搞郁闷了。RnD System公司的产品。我要测cell media中的
endothelin-1的含量。现在的问题是——standard老是做的很烂。我也不知道哪里出了
问题。我前后用了两个Kit，第一个Kit的standard没问题，但是第二个（我用这个做了2批）怎么做
standard都不行。
附件中是manual中给出的样品。把浓度取log10做X轴；Luminescence取log10做Y轴，应
该是线性。我做的线性非常差，而且低浓度的standard的deviation很大（duplicate）
1）会不会是第二个Kit的antibody公司coat的不好？或者质量有问题？
2）log后才线性，那这个kit是不是本身就很容易出问题？Assay Design有个Kit，是直
接用原始数据划线，就是线性。这个会不会更精准一点？我一直不喜欢4 parameter或者其他方式
处理原始数据后才线性的kit。
3）最后一步incubation（产生luminescence的步骤），manual上说要Incubate 5-20分钟，而
且说了，这15分钟内... 阅读全帖 |
|
V********n
发帖数: 305 | 32 理论上都能增加阳性程度，但也可能增加假阳性。俺的经验，供参考。
１）增加lysate，同时增加incubation volume（相当于保持蛋白浓度不变，或者降低
蛋白浓度），有助于降低non-specific
２）incubation时间不要过长，若干小时，最长过夜吧
３）最后清洗充分 |
|
l*****g
发帖数: 43 | 33 1，下马威，两周前-80掉电，东西都变象春天一样的很温暖
2.新的incubator，两周后发现是8.1，不是neuron啊
3.换一个incubator，两天后发现是30度，不是在表达蛋白啊，还有牛的细胞，牛的体
温是39，大家知不知道
好不容易几个customer，一世英名！
自暴自弃在网上玩儿，下午再干活儿吧！ |
|
b*********b
发帖数: 64 | 34 I use QIAGEN Plasmid Midi kit. 100ml culture could give about 30-50ug. I
tried Qiagen's large construct kit.But in my hand, the midi kit is much much
better than the large construct kit, and also takes much less time.The
following is the protocol I got from Qiagen.
User-Developed Protocol:
Isolation of BAC DNA using the QIAGEN® Plasmid Midi Kit
This procedure has been adapted by customers from the QIAGEN® Plasmid
Midi Kit Protocol.It has not been thoroughly tested and optimized by QIAG... 阅读全帖 |
|
b******r
发帖数: 111 | 35 Note: NHEs is membrane proteins that transmembrane 12 times. NHE5 is
localized to the plasma membrane;NHE6-9 reside on organellar membranes(
endosome,Golgi);
1. pcDNA transfects NHE5-9 through Fugene 6(ratio is 3 uL of Fugene/2 ug of
DNA) in 293 cells. After three days,collect cells.
2. Cold PBS washes cells. Add 150 uL of lysis buffer(final concentration:
50mM Tris pH7.4, 150mM NaCl, 1% triton, 0.1% sds, cocktail inhibitor)
into each well of 6-well plate. Scrape cells.
3. The lysate gets st... 阅读全帖 |
|
D*a
发帖数: 6830 | 36 Quick Tail Prep
Collect mice figure/tail in 0.5 ml Eppendorf tube. (4 mm is enough)
Add 100 microliter of quick tail digestion buffer (below).
Incubate at 55 C for 2 hours to overnight.
Vortex and incubate at 95 C for 10 minutes.
Centrifuge for 5 minutes to get rid of the small pieces of undigested sample.
(NOTE: I do neither vortex nor centifuge, and my pcr works
but if you vortex, you have to centifuge)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever ... 阅读全帖 |
|
h******y
发帖数: 351 | 37 这家公司也在卖水稻表达的人血浆白蛋白。
http://www.amsbio.com/Animal-free-Human-Serum-Albumin-HSA-ecoHS
上几张他们网页上的图给大家看看，行家说说这样的活性算不算好。
Purity and Size of Competitive HSA Products by SDS-PAGE
Figure 1 below shows a Coomassie blue stained gel (left) and a silver
nitrate stained gel (right) comparing size and content of competitive HSA
products. The figure shows that there is no difference in migration patterns
or purity between: 1) ecoHSA™ , 2) a commercial human plasma serum
albumin purchased as a lyophilized ... 阅读全帖 |
|
f*********e
发帖数: 1144 | 38 我想请教一下版上有Ab Ag binding经验的如下问题：
我的样品（Ag)里面有不同的species 都可以跟Ab 作用，因为它们都有相同的epitope.
比如，A, B,C,D 都可以跟Ab作用。但是[A]>[B]>[C]>[D]. 不知道到底浓度差别多大
，估计最大最小之间有20倍。
我先在的问题是，after incubation for two hours, A可以被capture, B也可以，但
是C和D都没有。所以我就在想是不是Ab的位点可能都被饱和了？有没有什么方法可以证
明确实是因为这个原因？比如after incubating with the real sample, 然后加入更
高纯度的已知Ag(E)with better affinity，看会不会再有这个E的出现？但是首先需要
确认的是这个binding是不是reversible,那个E会不会kick off previously captured
A and B?
多谢多谢！！！！！
|
|
m****M
发帖数: 360 | 39 After how long incubation did you get the colonies? It's not real bacteria
containing your plasmid, probably satellite colonies if you got them after
longer incubation. |
|
t****g
发帖数: 120 | 40 今天做shRNA lentivirus 的感染实验，在serum free medium里incubate了6个小时，
拿出来准备加含FBS的medium的时候不小心在hood里洒了含有病毒的medium.
两个问题：
1。安全。 我尽量用含bleach的喷液清洁了hood里洒了medium的地方，但还是害怕没清
干净。万一有残留在手套上的，然后再污染到hood外面别的地方，会有危险吗？
2。实验。 按照protocol细胞应该在有病毒的medium里培养48小时，然后开始
selection. 可我的这些细胞只incubate了6个小时，是不是会影响transduction
efficiency很多啊?
真够倒霉的！ |
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a****d
发帖数: 1919 | 41 Strategy in our lab：
Put all the suspicious cells in one quarantine incubator,using detection kit
like this to test your cells every month. Then move all myco-negative cells
to decontaminated incubator.
http://www.rndsystems.com/product_detail_objectname_MycoProbeMy
MycoProbe® Mycoplasma Detection Kit
It's always better to prevent spreading of Myco in the first place. |
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j****t
发帖数: 1663 | 42 我是在这个lysis buffer里作sonication的。我现在用0.2% sarkosyl（instead of 0.
5%）,和 1% of Na-deoxycholate。sonication的效果很好。而且这个浓度的detegent
对于IP 来说还是高了些，我会在做IP前把sample稀释一倍。
我觉得你的lysis和sonication 的条件也许可以了，问题说不定是在reverse-
crosslink。我的快速检测片段大小和ChIP DNA浓度的步骤如下，供你参考参考。
Check chromatin size and conc.
1. Dilute 50 ul chromatin extracts with 50 ul TE buffer. Add 4 ul of 5 M
NaCl and 1 ul of 10 mg/ml Proteinase K (Invitrogen #25530-015) (use a PCR
tube).
2. Incubate at 65 oC for at lease 4 hr (using a PCR machine) ... 阅读全帖 |
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p*l
发帖数: 1359 | 43 CO2 incubator是必须的，不可能自己把非CO2 incubator 改装成CO2的。新的不便宜，
想省钱的话叫老板买个二手的。 |
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D*a
发帖数: 6830 | 44 Quick Tail Prep
Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
Eppendorf tube.
Add 100 microliter of quick tail digestion buffer (below).
Incubate @ 56 C several hours to overnight.
Vortex and incubate @ 99 C, 10 minutes.
Centrifuge @ max speed, 5 minutes.
(NB i don't vortex so i don't need to centifuge the tube, it works)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever you routinely do).
Transfer supernatant to a fresh tube for s... 阅读全帖 |
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D*a
发帖数: 6830 | 45 Quick Tail Prep
Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
Eppendorf tube.
Add 100 microliter of quick tail digestion buffer (below).
Incubate @ 56 C several hours to overnight.
Vortex and incubate @ 99 C, 10 minutes.
Centrifuge @ max speed, 5 minutes.
(NB i don't vortex so i don't need to centifuge the tube, it works)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever you routinely do).
Transfer supernatant to a fresh tube for s... 阅读全帖 |
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h*******o
发帖数: 4884 | 46 1) try other blocking solution and block longer
try BSA, non-fat milk, or both and block at least for 1 hour
2) Reduce your 2nd antibody concentration.
3) For primary antibody incubation, incubate in blocking buffer and try
either 1-2 hr at RT or 4oc overnight, whichever one you haven't tried yet |
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l**********1
发帖数: 5204 | 47 RE 孵蛋的学弟/妹 giantbird05 (大鸟)
of course you can do it, just Store it in freezer.
pls refer:
Gelatin/Fibronectin
• Place 0.1g gelatin into a 500 ml glass bottle. Add 500 mL of dH20
and autoclave. The
concentration of gelatin is 0.02%. Fibronectin comes as a 5 mL liquid.
Dilute 1 mL of
fibronectin in 80 mL of 0.02% gelatin in a T-75 flask. Invert to mix.
Immediately
aliquot into 8 tubes of 10 mL. Store in freezer.
Coating Flasks
• All flasks must be coated with gelatin/fibronectin the ... 阅读全帖 |
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l**********1
发帖数: 5204 | 48 RE LZ:
pls try
Cassette-ligation downstream 3'RACE PCR
or Tail-PCR
Protocol:
看图说话
>
Amplification of FSTs by 3’-RACE
Total RNA was isolated using hot-phenol extraction
[27], from 20 ml cultures in 50 ml Falcon tubes on a rotating
wheel. Reverse transcription used the SuperScript
III First-Strand kit from Invitrogen in a DNA Engine
PCR machine (MJ research). Primer QT was mixed with
5 μg RNA, incubated at 65 °C for 5 min then cooled to
55 °C over 100 sec, after which the annealed mixture
was kept... 阅读全帖 |
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