c*****n 发帖数: 233 | 1 I washed a 0.1% DEPC solution bottle and I think I inhaled a little bit
because I smelled it. Is it a serious problem? Is there anything I can do? |
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n*******e 发帖数: 27 | 2 我的体会是只要你不向tube里吐口水,一般没有那麽厉害,用不着谈虎色变.
分离RNA的试剂里多有强变性剂如胍盐,酚等,所以这种情况下RNase也就多
被变性失活的说.最要紧的是你的储备溶液,用于溶解RNA的水,T.E., 用于
NORTHERN的STOCK等,要保持干净.玻璃器皿可在200C左右烤三小时再用.RNA
专用的药品,试剂要分藏.水用DEPC处理O/N,STIR,灭菌20MIN.TRIS要和DEPC
反应,所以要在灭菌破坏DEPC后加入.DEPC强烈致爱,你就自己看着办吧.
RNasin是RNase的抑制蛋白,但温度升高后会解离.
所有的操作要戴手套,小心谨慎.所有的试剂都要分装使用,用过一段时间就
扔掉.一般公司的试剂和用具都比较可信. |
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l**********1 发帖数: 5204 | 3 LZ
pls refer below english protocol forum their replied:
Dodger on Tue Nov 23 00: 2010 said:
It might be a little late to post an answer to this thread due to it's age,
but since it's pinned...
We usually prepare 4x 50 mL tubes. Fill three with 40 mL DEPC treated water,
and one with 100% ethanol.
One of the DEPC treated water tubes gets 5-10 squirts of RNase Zap.
Between each sample, the homogenizer is dipped into 100% ethanol, RNase Zap
water, and 2x DEPC treated water in turn.
Each time turn... 阅读全帖 |
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x**e 发帖数: 163 | 4 我一直用DEPC处理的FAA来固定植物材料,有一次石蜡切片是请别的实验室切的,所有
试剂都没有用DEPC处理,硬着头皮做完了ISH,结果和以前RNAse free的没什么区别。
当然ISH的试剂都是DEPC处理过的。似乎切片过程不太重要,而且材料在石蜡中保存半
年再做ISH也没有问题。 |
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l********s 发帖数: 70 | 5 最近做RNA从plate中提取,发现RNA测试有机溶剂那个参数很低,0.3-0.6 。 其实最后
我尽量把乙醇抽吸干净。我怀疑是不是DEPC水有问题,因为我统一吧RNA dilute 到
100ng/ul 做反转,发现dilute后,那个比值更低了。。。
当然Nanodrop 的control 用的是DEPC,这个不会搞错。。
不知道为啥,怎么提高这个比值? 另外这个如果低多大程度影响反转? |
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c*********d 发帖数: 9770 | 7 荒猫牛不耕微信号laoniu003005
功能介绍
好文分享
来源:荒猫之舞
作者:荒猫
中美贸易战告一段落,毫不意外地,咱们又赢了。不过与以往不同的是,这次美国也赢
了,是双赢。据报道,咱们答应了美国减少贸易逆差的要求,所以美国赢了;而因为要
减少贸易逆差,中方将大量增加自美购买商品和服务,这样可以满足咱们不断增长的消
费需求和促进高质量经济发展,所以咱们也赢了。
但让我感觉不可思议的是,这么好的事情,为什么一直不做呢?为什么要在美国政府的
逼迫下去做呢?是因为没有想到?那岂不是说那些人能力低下?是因为想到了而不做,
那岂不是证明了那些人很坏?
而且在美国高调启动贸易战之初,咱们是抱着多大的决心,宁为玉碎不为瓦全,一定要
打赢的,一定要粉碎美国人阻挡咱们大国崛起的阴谋的。当美国提出500亿的清单时,
咱们针锋相对;当美国提出1000亿的清单时,咱们继续应战;而当美国把清单的总价提
高到2000亿的时候,一夜之间,美国就不是阻挡咱们大国崛起,而是有利于咱们大国崛
起了。美国的要求原来是有利于咱们的发展,而不是遏制咱们的发展了。我恍然大悟:
原来以前的针锋相对,是用的激将法,是为了刺激美... 阅读全帖 |
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M****e 发帖数: 70 | 8 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 |
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M****e 发帖数: 70 | 9 make sure that you are aware of the buffer effect:
It is estimated that DNA has 15% lower absorbance in TE
buffer, 23% lower in TE+saline compared to water.
1 O.D.260 are 38, 45 and 50 ug/ml for DNA in ddH2O,
TE and TES, respectively. I assume the same for RNA,
and 1 O.D.260 are 40 ug/ml for RNA in TE (from Ambion).
btw, A260/A280 is different for pure RNA
DEPC'd H2O (pH5-6) 1.60
Nuclease free H2O (pH6-7) 1.85
TE (pH8.0) 2.14 |
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j******9 发帖数: 180 | 10 polyaerylamide DMSO SDS DEPC 二乙基焦碳酸酯 DTT TEMED 氯仿 Triton X
-100 等都用 只有DNA荧光染料类的东西让我有感觉 |
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v***a 发帖数: 1242 | 11 我也挑我知道的说说。
liver里提取RNA我也出现相同的状况,非常难溶,一般我会根据pellet大小加多点DEPC
水,如果还是不溶,我也不管它,貌似放到-80度储存后它最终会自己溶掉的。要么我
就多vortex一下。
有个数据说1mg的tissue中,muscle或brain等大约可以得到1-1.5 ug的RNA,而liver可
以得到6-10 ug的RNA。所以巨多RNA正常。 |
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w******a 发帖数: 1527 | 12 Check this one:
Reagents
* TRIzol from Invitrogen #15596-108.
* Mini RNeasy plus kit from Qiagen #74134.
* Phase lock gel from Eppendorf #2302830.
* Keep TRIzol at 4C.
* Use RNase-free tubes and tips for all steps.
* Prepare 70% ethanol using DEPC-H2O and keep it at -20C.
RNeasy plus kit contains a genomic DNA elimination column. It can replace
the DNase I digestion step of regular RNeasy kit.
1. Add 1.2 ml TRIzol reagent to cells/tissues (5~10X 106 cells/ml or 50~100
mg ... 阅读全帖 |
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b*********l 发帖数: 490 | 13 I am thinking another issue. During the process of zebrafish like fixation-
dehydration-rehydration, all of the MeOH+PBT solution should be with DEPC
treated water or what? Is it possible that the mRNAs in zebrafish have been
deteriated before the hybridizaion? I am not sure how bad the
invivo mRNA will be if I don't use RNase free reagents. The thing is I
couldn't see any difference between negative control and the real samples.
Thank you for any suggestions. |
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a******n 发帖数: 392 | 14 根据我做了几年的 in situ 的经验,不需要DEPC处理的水,任何一个步骤都不需要。4
% PFA 固定24小时,样品里的
mRNA不用担心被降解。
been |
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a********k 发帖数: 2273 | 15 it is true when you get a result...but it is not true when you are dealing
with some low-expression genes. I got results from both, but I always use
DEPC water later on.
。4 |
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s******r 发帖数: 2876 | 16 那冰冻组织和福尔马林fix的组织,哪一个更适合做in situ,
请问各有什么利弊?
根据我做了几年的 in situ 的经验,不需要DEPC处理的水,任何一个步骤都不需要。4
% PFA 固定24小时,样品里的
mRNA不用担心被降解。
been |
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h******y 发帖数: 351 | 17 I asked the same question before on Histonet and other forum but never got a
satisfactory answer. :-(
I was doing in situ hybridization using TBS or PBS and came across another
protocol using Maleic acid. Then I went back and checked the data sheet
coming with the anti-DIG-AP antibody (Roche), and found that they suggested
using Maleic acid based buffer for "membrane application" and Tris-based
buffer for "other application".
Maleic acid has less buffering capacity than Tris in the pH range of 7... 阅读全帖 |
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v***a 发帖数: 1242 | 18 要配一个200 mM MOPS buffer,做RNA的相关实验,如果我用DEPC-treated water来配
,在配好后还需要拿去autoclave吗?谢谢~ |
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f*******e 发帖数: 628 | 19 用过 DEPC 水,没问题的,除非没有处理好。
还是提纯时候有机溶剂没除干净吧? |
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l********s 发帖数: 70 | 20 可能找到原因了,确实是DEPC水出的问题。 谢谢楼上兄弟。我索性用水稀释算了,反
正马上反转 |
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m*****y 发帖数: 857 | 21 just curious, how do you even prepare RNase free water? Don't tell me
DEPC.....
water, |
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c*****n 发帖数: 233 | 23 yield非常低(5oug/ml)怎么办?
A260/A280 在1.5-2.0之间。mRNA用DEPC treated water稀释过。 |
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C*****h 发帖数: 926 | 25 you will die.
80 years later. |
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l**********1 发帖数: 5204 | 26 For manipulating single strand RNA, LZ should use RNase free DEPC water for
the preparation of that 70% Ethanol. |
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b****r 发帖数: 17995 | 27 看了半天没有一个人说
RNA 和DNA降解后,变成短链甚至核苷酸,260都是升高的,260:280甚至会高于2
如果你根据这个读书觉得质量“超级好”,那你就要小心了
简单的用DEPC H20 做胶和buffer,洗干净tank跑一下就知道了,大概1k多以下应该有
两条带,分别对应两条核糖体RNA,没有的话就可以歇了 |
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v**********t 发帖数: 9 | 28 从Dharmacon订的RNA,25mer, single-strand, HPLC purified, deprotected,
desalted.
拿到手后用DEPC water resuspend, 加到蛋白溶液后就把蛋白给沉淀了。先后订的三
批RNA全这样。
哪位知道这是怎么回事? |
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