j******2
发帖数: 544 | 1 昨晚离开实验室前配了media，用了3.5支FBS，并在实验室白板上写明了，需要再
aliquot。由于配media取用FBS的时候正好被T看到，我当时心就抽了一下，有不详的预
感。T是负责aliquot FBS的人。晚上收到T的邮件，发给实验室所有人，如下：
People,
We seriously need to work on communicating more effectively on things that
need thawing time and aliquoting, like the FBS. It just so happened that I
looked yesterday before I left the lab at the FBS aliquots and we had 5. I
took out FBS to thaw this morning and right now at 6pm we are down to 1
aliquot. We are going through medium like crazy, which means ... 阅读全帖 |
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g*********5
发帖数: 2533 | 2 Isolation of nuclei for the co-IP and whole ChIP protocols is based on the
methods of Gendrel et al. (2002, 2005), Johnson et al. (2002), and Nelson et
al. (2006).
METHOD
For extraction and co-IP of nuclear proteins, see Steps 1-39 (Fig. 1). For
the ChIP procedure, see Steps 40-69 (Fig. 2).
Figure 1.
View larger version:
In this page
In a new window
Figure 1.
Flowchart for the timeline and organization for co-IP of nuclear proteins
from Arabidopsis seedlings.
Figure 2.
View larger versio... 阅读全帖 |
|
j********r
发帖数: 156 | 3 As to 3MA, it is hard to dissolve it. The following is the method I always
use to prepare 200 mM stock in water:
For the Sigma 3MA (M9281-100MG)
1. Heat a large amount of tap water (say 500-1000 ml) to around 70 celsius
degrees.
2. Heat 3.352 ml tissue culture-grade water in a tube (eg. the tube for
flow cytometry or the regular 15 ml conical tube), by sitting it
in the heated water.
3. Remove the whole stuff into a hood (the temp of the tap water can
sustain for a while w/o a heater... 阅读全帖 |
|
h*******o
发帖数: 4884 | 4 Unless the antibody comes as lyophilized powder, it is recommended to store
the anitbody in un-diluted aliquots. Diluted antibody is not as stable as
the original stock.
For lyophilized powder, usually add PBS or ddH2O following the product data
sheet, then aliquot for storage. |
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q*****d
发帖数: 445 | 5 最近要做library, 使用这个实验室的方法进行易错PCR
（http://www.msg.ucsf.edu/agard/Protocols/PCR_Random_Mutagenesis.htm），酶
是NEB的Taq,但是效果一直不是很好，35 cycle PCR的浓度还是很低，30cycle特别的少
，请问
大家有什么方法提高产量吗？还是我的方法不够好，谢谢大家。
PCR Random Mutagenesis
Materials
1. Parental plasmid
2. Oligos surrounding region to be mutagenized
3. Error-prone PCR buffer (10x is 100mM Tris-HCl, pH8.3; 500mM KCl,
70mM MgCl2, 0.1% (w/v) gelatin.)
4. 10mM MnCl2 (stock) (make sure there is no brown coloring to
stock soln; if there is it means... 阅读全帖 |
|
b*********b
发帖数: 64 | 6 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 |
|
l**********1
发帖数: 5204 | 7 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********k
发帖数: 14844 | 8 得多熟练的千老才能在0小时内把1000瓶水aliquot好再重新编码混合? |
|
|
|
C*I
发帖数: 4736 | 10 一直在误导，现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人，必须
经过一个中间宿体性的其它动物才能传染给人。所以，病毒发生后，就故意误导全国人
民去海鲜市场找证据，找其它野生动物的麻烦。 而且还是病毒所去找的，找完了还装
模做样化验呀，分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物
，一直不敢说，说了其它相关已经在人就会去早那种动物监测。 所以压根不说，打马
虎眼。
事实上，早在2013 年，就是这个武汉病毒研究所，已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒，其中就包含了类
似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体，并能够感
染人的细胞。对此新发现，武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1
”，以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载
于2013年11月的《自然》杂志。
就是说，从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus， 可以不经过
其它受体/宿体，而直接传染给人。 他们... 阅读全帖 |
|
C*I
发帖数: 4736 | 11 一直在误导，现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人，必须
经过一个中间宿体性的其它动物才能传染给人。所以，病毒发生后，就故意误导全国人
民去海鲜市场找证据，找其它野生动物的麻烦。 而且还是病毒所去找的，找完了还装
模做样化验呀，分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物
，一直不敢说，说了其它相关已经在人就会去早那种动物监测。 所以压根不说，打马
虎眼。
事实上，早在2013 年，就是这个武汉病毒研究所，已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒，其中就包含了类
似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体，并能够感
染人的细胞。对此新发现，武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1
”，以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载
于2013年11月的《自然》杂志。
就是说，从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus， 可以不经过
其它受体/宿体，而直接传染给人。 他们... 阅读全帖 |
|
C*I
发帖数: 4736 | 12 Published: 30 October 2013
Isolation and characterization of a bat SARS-like coronavirus that uses the
ACE2 receptor
Xing-Yi Ge, Jia-Lu Li, Xing-Lou Yang, Aleksei A. Chmura, Guangjian Zhu,
Jonathan H. Epstein, Jonna K. Mazet, Ben Hu, Wei Zhang, Cheng Peng, Yu-Ji
Zhang, Chu-Ming Luo, Bing Tan, Ning Wang, Yan Zhu, Gary Crameri, Shu-Yi
Zhang, Lin-Fa Wang, Peter Daszak & Zheng-Li Shi
Nature volume 503, pages535–538(2013)Cite this article
Abstract
The 2002–3 pandemic caused by severe acute respirator... 阅读全帖 |
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j****9
发帖数: 50 | 13 I save them. Since we make home-made solid food for my 8 month old baby now,
I aliquot the solid food into those bottles and freeze them. I feel those
glass bottles is way better than plastic one. |
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j***a
发帖数: 10844 | 14 Sounds scientific! And good description.
How about take a small amount of aliquot. Filter it and then wash it out and count under a microscope? |
|
s***l
发帖数: 42 | 15 多做一些aliquot, 藏在-80度，有一套专门的buffer用于作RNA的实验，包括乙醇，
甲醇什么的，坚决不能用于DNA的实验。【 在 yzhanc (子曰) 的大作中提到: 】
窍 |
|
k**e
发帖数: 2728 | 16 en?
非极性溶剂是destablize nucleotides的啊。我没有做过跟RNA相关的实验，但是RNA是
应该避免碱性条件，至于用水还是用乙醇，我认为用水好点。因为水是极性的分子，可以
稳定RNA的结构
还有nucleotides之类的东西，以及NTP、dNTPs啦等溶液的储存，应该是根据自己每次实验
大概需要的量，aliquot到separate tubes里头。每次实验只拿出一个tube来解冻，争取1
次到2次的实验就能把那个tube的样品用完。 |
|
p**o
发帖数: 3409 | 17 不知道版上有没有用Python做科学计算的同好，
本人近日在带Lion的新MBA上安装Python科学计算包遇到了困难，
现把失败经历小结如下，希望后来人不要重走弯路，
如果能为本人指出一条“正路”就更感谢。
尝试1. MacPorts
macports是我第一个试的，下面这些包可以通过编译：gnuplot libsvm py27-numpy
py27-scipy py27-matplotlib python27-doc py27-sympy py27-ipython py27-
networkx py27-pymc py27-django py27-libdnet py27-lint py27-mysql py27-pip
py27-ply py27-pygraphviz py27-pyrex py27-rpy2 py27-scientific py27-sqlite
py27-svn py27-tables py27-tornado igraph py27-igraph py27-numexpr py27-pp
py27-cython py27-psyco py27-twisted... 阅读全帖 |
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n*******e
发帖数: 27 | 18 biz is a master guy on pcr. i have a little to say. i have been using
tail DNA for pcr characterization of mice KO. my experience is that
u should separate all the reagents/pippets/pippetmen from commonly
used stuff. so, first of all, go to clean the pcr station, and make it
as clean as possible. remember, always NOT to bring template to the
station. also, get pcr buffer/ddH2O changed frequently if u question it.
primers should be stocked as 100uM, and u may only aliquot them for use.
use filter |
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M****e
发帖数: 70 | 19 the essential step is to keep everything as clean as possible.
and do not use anything you are suspicious. Ambion is a good
company that has very good experience dealing with RNA. my
personal experience is that: ALWAYS aliquot reagents. i used
to use DEPC'd ddH2O, but now i would rather use commercial
nuclease free water. try to be a quick and clean hand, quick
and dead, you know that? i.e., you quick and RNase dead, no
no vice versa. when you deal with different biological samples,
you have dif |
|
f*********e
发帖数: 1144 | 20 Starting: total 1mg whole cell lysate
After glycoprotein enrichment, no idea.
Assume: 100 glycoprotein obtained after pull-down and total amount is 1% of
total whole cell lysate, so each glycoprotein would be 100ng. Then split
into 3 aliquots and reconstitue in 30ul, so around 30ng for each protein at
the concentration of 1ng/ul. My estimation is actually lower, given the
target protein is low abuntant. |
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y******n
发帖数: 9 | 21 有些抗体是不能直接冻的。要价Glycerol或其他。要Aliquot成很多小管，每次取一管。 |
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y******n
发帖数: 9 | 22 有些抗体是不能直接冻的。要价Glycerol或其他。要Aliquot成很多小管，每次取一管。 |
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g*********5
发帖数: 2533 | 23 you could try express myc-A, AND Aliquot cell lysate then use myc-antibody
or corresponding igG as control?
时候
negative
speci
pr |
|
K******S
发帖数: 10109 | 24 don't worry, peptides are VERY stable. especially your peptide, I can't
think of any proteolytic enzyme that can cleave the sequence.
If you can, just make some aliquots |
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c*****e
发帖数: 436 | 25 thank you so much! what is aliquots, by the way? |
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h***l
发帖数: 168 | 26 you need to thaw it at 65. It is not absolute, clear solution, so you need
to "reconstitute" at 65 then aliquot. |
|
p****y
发帖数: 95 | 27 PEI Transfection
Transfection:
1. Split 293T cells one day before transfection in DMEM/10% FBS medium:
a. 6 well dish: 0.5x106 cells
b. 10cm dish: 4.0x106 cells
c. 15cm dish: 9.0x106 cells
2. Prior to transfection bring all reagents to room temperature.
3. In a sterile tube dilute total plasmid DNA (ug) in serum-free DMEM w/o
phenol red (volume of media is 10% of final volume in culture vessel). Use
transgene: viral packaging (psPAX2):viral envelope (pMD2G) constructs at 4:2
a.... 阅读全帖 |
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s******y
发帖数: 220 | 28 磷酸化的WB，上次看有带，这次没了，怀疑是phosphatase inhibitor cocktail 坏了。
我用的是calbiochem 的Phosphatase Inhibitor Cocktail Set II，
lypholized powder溶解后aliquot出来很多小管，store在-20，用的时候拿出来放4C不
放回去，
这样容易坏吗？
btw， 请推荐好用的phosphatase inhibitor cocktail,谢拉。 |
|
h*******o
发帖数: 4884 | 29 Abcam几年前还勉强可以
最近2年明显有Santa Cruz化的趋势，而且经常缺斤少两。100ul的抗体，我aliquot只
有80多一点
我现在是只要其他口碑还不错的公司有的抗体，坚决不从Abcam和Santa Cruz买 |
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D*a
发帖数: 6830 | 30 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 |
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o*****r
发帖数: 156 | 31 EDC and NHS are aliquoted and stored at -20, every time when you need to
activate the CM chip, thaw both of them, mix and inject over the surface (I
usually use 5 ul/min for 7 min).
http://cspr.uthscsa.edu/activation.php |
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p*****m
发帖数: 7030 | 32 不知道，是从公司要来的半旧library,你可以去周围的screen center打听下，他们卖
不卖aliquot,一般这样几K就搞定了 |
|
M*****n
发帖数: 16729 | 33 这个用DMSO配成多少浓度的stock solution？
要不要aliquote 之后freeze at -20?
反复冻融是不是不好？
在室温下，rapamycin是不是不稳定?
谢谢 |
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v*********d
发帖数: 382 | 34 DMSO 比ethanol 好，50mM stock，aliquote，－20
其实还算比较稳定的 |
|
V***b
发帖数: 3419 | 35 20 nM is more than enough to inhibit TORC1. Stock solution 20 uM in DMSO;
make small aliquote and keep in -80; freeze-thaw cycle does not severely
impair its function, but not recommended. |
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s******a
发帖数: 472 | 36 http://ge.geglobalresearch.com/blog/you-know-youre-a-biologist-
This list has been floating around the internet for a while now but it is
too funny and too true not to pass along. I am not sure where the original
came from, so if you happen to know, please let us know in the comments so
we can give them credit!
You know you’re a biologist when…
You regularly open the toothpaste with one hand.
You wash your hands before and after using the washroom.
You’re looking for a cook book by Maniatis
When... 阅读全帖 |
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q*****n
发帖数: 331 | 37 Many antibodies (which Abcam sells) are not produced by Abcam.Abcam, and
several others, are more like dealers. They buy bulk antibodies from other
vendors, and re-sell them in small aliquots to make money. Their insert
figures could be from another company. For instance, one antibody was made
by a very small company, and several vendors including Abcam purchase bulk
amount and re-sell it. The initial quality testing data was provided by that
small company. That is why you see the same insert fi... 阅读全帖 |
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g*********9
发帖数: 3528 | 38 You could just suggest to aliquote more fcs since your lab Is using fcs
crazy.
Key point: keep research going is the priority.
Lab manegement should work FOR it.
Then next time, you could hide things as well....since everyone else in your
lab is doing this, they must have their reasons.
Suggest: Watch Dowton Abbey, learn how to communicate in English.
that
I
I
items |
|
s******s
发帖数: 13035 | 39 担心就再chloroform抽一下， rneasy。我估计没问题，260/230这个指标
有时候我用机器做，同样的aliquot会差很多
qPCR |
|
D*a
发帖数: 6830 | 40 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 |
|
D*a
发帖数: 6830 | 41 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 |
|
F*****e
发帖数: 182 | 42 1. 100mg tamoxifen in 1ml 100% ethanol, vortex for 2-5mins.
2. Add sesame oil to 10ml, leave it on a shaker until fully dissolved.
Aliquot into 500ul/tube and store tubes in -20oC for 1-6 months (final
concentration: 1mg/100ul).
3. Oral administration: 250-350ul/25g body weight. |
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F*****e
发帖数: 182 | 43 1. 100mg tamoxifen in 1ml 100% ethanol, vortex for 2-5mins.
2. Add sesame oil to 10ml, leave it on a shaker until fully dissolved.
Aliquot into 500ul/tube and store tubes in -20oC for 1-6 months (final
concentration: 1mg/100ul).
3. Oral administration: 250-350ul/25g body weight. |
|
h*******o
发帖数: 4884 | 44 Seriously?
You need to learn how to turn people down.
First of all,you should never let him know that you have all he wants in the
first place.
Pretend that you don't have any left.
For antibodies, make aliquots and store in your own place.
If he asks for it, tell him that you are on your last vial and just keep re-
using a diluted tube. Tell him that you can share with him the re-used tube
but he has to return it so that you can continue to use it.
Nothing he can complain about.
box |
|
p*****2
发帖数: 12 | 45 There is no lab around you working on rice? You can ask an aliquot of rice
cDNA from any lab who working on rice. As xihe said, you should specify
which rice variety you need. We have cDNA of several varieties, but not sure
if there is one you need. But I think you may need cDNA of the reference
variety, Nipponbare. |
|
G***y
发帖数: 1082 | 46 Aliquot out enough mastermix for one plate only, then add mastermix to a
new plate before adding template. In that case I just use one set of tips
for the mastermix. |
|
i***0
发帖数: 160 | 47 If it is an enzyme, each thaw and freeze cycle will reduce its activity. If
IgG or other immunoproteins, you'd better thaw the sample and separate in 20
uL aliquotes. Freeze thaw is not good for most proteins, mostly because it
might disrupt protein folding. |
|
b****s
发帖数: 148 | 48 Ab is less likely to go wrong if you can have signals from other probings.
your sample prep might be the target of troubleshooting.
1) did you freeze and thaw your sample frequently? - if yes, your current
samples are no longer reliable. make aliquotes in the future, or boil
protein right after lysis.
2) must snap freeze and thaw on ice for all ampules
3) double check your lysis process and use new protein samples -
microtubules can be very hard to dissolve or remain soluble |
|
p*****m
发帖数: 7030 | 49 没那么夸张了。你们Mcknight搞老鼠的当然要资源又risky,用fly/fish做个这个screen
花不了几个钱的 构建一个inducible cell ablation line,然后养出来招个tech往每个
管子/well里面加药就行。如果嫌看imaging太麻烦，还可以直接kill掉能产生行为特征
的细胞,这样看一眼就知道drug有没有用了。这个是我强项 哈哈哈
我前一段做了个1000-drug的fish screen,挺容易的。。我前一段联系了几个screen
center,有人愿意把用剩下的drug aliquot卖给我，1万个drug才要三四千刀，市场价要
五六万 哈哈 不过我还没买 暂时没法做
risky
a
support
risky |
|
p*****m
发帖数: 7030 | 50 很多 fish说实话 我的感觉是不做drug screen没啥太大的前途。
库有好多种，从最小的比如fda drug lib几百到一千，到已知target的bioactive什么
的四五千，然后到一两万的都有。再多的一般就不是一个单独的lab能screen得了的了
，药厂的lib上百万都不奇怪。
用几回当然得看物种，fish的话，一个commercial lib够用上百次了。。所以我才到处
找那里能卖给我点用剩的aliquot。先做细胞也不错，细胞的问题就是你要知道药厂都
在做cell based screen/biochem based screen,你能想出来的assay他们八成早就筛过了
其实说到底，还是得有个好assay，有的话后面不是问题 |
|
