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cy3 cy5的标记

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楼主

铁杆站友

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这个帖子发布于13年零107天前，其中的信息可能已发生改变或有所发展。

今天标记cy3/5，像往常一样用pcr机器作，37度3小时。我们的机器一个面板控制两个block，就是可以同是做两种条件。我当时摸了一下block A,前面刚有人用过，很烫手，所以就采用了block B, 结果面板设置给调到A上了，意思就是样品放到B里面，但是程序没有加上，室温了将近1个半小时，我才发现。赶紧把程序加上，37度3小时。结果刚才纯化DNA，去测了下标记率，低得惨不忍睹，几乎可以说没有标记上。

以上的愚蠢错误，今后一定会注意，也算是个反面教材提醒大家吧

我想问问，低的原因仅仅是室温放置时间过长导致吗（室温1各半小时，然后37度3小时）？会不会是dye也不是很好了（今年新7月底买的，一直避光4度保存）？

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2007-10-11 06:08 浏览 : 7943 回复 : 2

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nkriver

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7月底的染料应该没有什么问题吧，不过我们的染料是在－20避光保存的（agilent）

2007-10-11 10:24

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huangglen

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先说明下，本人是新手，一次实验都没做过，在此说说我的看法而已。

要知道楼主的答案，应该只要重复一次标记，按正常操作（短时间，合适温度）就可以知道了。

另外，看到一个protocol（3DNA Array900 miRNA Direct）上有对减少Cy5降解的描述，在此摘下来：

Cy5 dye performance may be affected by a variety of factors that are particularly prevalent during the summer
months. Exposure of the Cy5 dye solutions and the hybridized arrays to light and to oxidative environments
may cause rapid fading of the Cy5 dye, regardless of the labeling system used. Limiting or controlling the
exposure of the arrays to these environments has been shown to significantly reduce Cy5 fading. Below are
recommendations for reducing the degradation of Cy5 when performing microarray experiments:
1. Always keep solutions and arrays containing Cy5 away from light, particularly sunlight. Cy5 will photobleach
when exposed to light, including normal fluorescent lighting.
2. Protect the hybridized, dried array from contact with air, particularly on hot and sunny days. We have found
that ambient ozone levels resulting from summertime air pollution can cause oxidative degradation Cy5.
Keeping the Cy5-containing arrays in an inert atmosphere (nitrogen) in a small container (50mL tube) can
significantly delay fading of the Cy5. Some investigators also add small quantities of dithiothreotol (DTT) or
beta mercapto-ethanol (BME) to the bottom of the tube to further promote a reducing micro-environment.
Be certain to avoid contact of the array with these chemicals. If possible, do not place the arrays or the
array scanner near high ozone-generating equipment, such as a freezer or laser printer.
3. In the preparation of wash buffers, avoid the use of water that may damage Cy5. As noted in the Internet
List Serve, MilliQ® water has been shown to damage Cy5 (http://groups.yahoo.com/group/microarray/
messages/2867). We recommend using deionized, reagent grade water from VWR (Cat. No. RC91505) to
prepare wash buffers.
4. In the preparation of wash buffers, be certain that any DEPC treated solutions have had all of the DEPC
fully removed, since DEPC is a potent oxidizer. We recommend using 10% SDS from Ambion (Cat. No.
9823) and 20X SSC from Ambion (Cat. No. 9763) to prepare wash buffers.
5. Add a small quantity of dithiothreotol (DTT) to the first two wash buffers used after the 3DNA hybridization.
The DTT should be at a final concentration of 0.1-0.5mM. Be sure to work with fresh DTT, as old or poor
quality DTT may cause an increase in background visible as a “haze” in the Cy3 channel.
6. Use DyeSaver 2 (Genisphere Cat. No. Q500500) after the final washing and drying of the microarray.
DyeSaver 2 is easy to use, compatible with most array surface chemistries, and protects Cy5 from
atmospheric oxidation for at least three weeks. DyeSaver 2 has also been shown to reduce Cy5 damage due
to photo-bleaching. For more information about DyeSaver 2, please refer to the Genisphere web site
(www.genisphere.com) or call Genisphere Technical Support.

2007-10-11 19:51

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楼主

凋亡2

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nkriver
7月底的染料应该没有什么问题吧，不过我们的染料是在－20避光保存的（agilent）

你好，我的染料是标明4度，几个月前新定的染料，被同事误放到-20，结果标记不上，后来换了一批就标上了。可能储存条件也与公司有关，我那个是perkin elmer的。

我有点担心，放燃料的那个冰箱门总是会关不严。

2007-10-11 21:54

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