w******e 发帖数: 1187 | 1 P32衰变后变成S32,那么衰变后的产物可能被T4 kinase incorporate
到5'END of DNA/RNA吗?如果会,那么放置了一两个half life后的
P32-ATP用于labeling后岂不是导致大部分product都不hot?
请牛人指点。多谢! |
o**i 发帖数: 1165 | 2 p32 半衰14天 2个28天
我们lab从来不用7天以上的
【在 w******e 的大作中提到】 : P32衰变后变成S32,那么衰变后的产物可能被T4 kinase incorporate : 到5'END of DNA/RNA吗?如果会,那么放置了一两个half life后的 : P32-ATP用于labeling后岂不是导致大部分product都不hot? : 请牛人指点。多谢!
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k******e 发帖数: 8870 | 3 carefully plan your experiments and use the P32 right the days right after
it comes in. |
w******e 发帖数: 1187 | 4 多谢两位的回复。Just to confirm: you guys actually just use the P32 for
a few experiments and throw it away? Our lab is really new to this
radiolabeling business, and we only do it intermittantly. It would seem
a big waste if I order a package and only use a few ul and throw the
rest away... any suggestions?
Thanks!
【在 k******e 的大作中提到】 : carefully plan your experiments and use the P32 right the days right after : it comes in.
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o**i 发帖数: 1165 | 5 1 p32 ATP 不贵
2 如果你从来没做过 还是去一个做过的lab培训一下
p32的能量中等 spill了就麻烦了
【在 w******e 的大作中提到】 : 多谢两位的回复。Just to confirm: you guys actually just use the P32 for : a few experiments and throw it away? Our lab is really new to this : radiolabeling business, and we only do it intermittantly. It would seem : a big waste if I order a package and only use a few ul and throw the : rest away... any suggestions? : Thanks!
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a***e 发帖数: 1010 | 6 occasionally, I use P32-ATP even after 1 month.
It is still hot enough for most of the experiments. |
l*******k 发帖数: 361 | 7 1 如果是gamma-32P ATP, 理论上还是可以被incoporate的,alpha-32P ATP应该就不行了 (这个不确定,说错了不要笑话我)其实买到的都是hot/cold ATP的混合物,放时间长一些,就是hot/cold的ratio降低了
2 放了一个月的还可以用,不过效率低了很多,最好用fresh的,我一般用14天之内的
,每个瓶子上都会标注时间
3 一瓶50微升的大概是六七十块钱吧,不是很贵 |
w******e 发帖数: 1187 | 8 thanks to all! I'm going to use gamma-32p ATP to label the 5' end of my RNA.
it's a pain to get the RNA so I'd hope to get as high a radiolabeling
efficiency as possible. Now I think the best option for me is probably
use the freshest stock possible.
one more dumb question:
I heard from my colleague that they got "hotter"product by using a 10mCi
stock then a 250uCi stock. But that doesn't make much sense to me, as I
assume
one can only label one P32 to one RNA sequence, so if the kinase efficien
【在 l*******k 的大作中提到】 : 1 如果是gamma-32P ATP, 理论上还是可以被incoporate的,alpha-32P ATP应该就不行了 (这个不确定,说错了不要笑话我)其实买到的都是hot/cold ATP的混合物,放时间长一些,就是hot/cold的ratio降低了 : 2 放了一个月的还可以用,不过效率低了很多,最好用fresh的,我一般用14天之内的 : ,每个瓶子上都会标注时间 : 3 一瓶50微升的大概是六七十块钱吧,不是很贵
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O******e 发帖数: 4845 | 9 哪个公司的gamma-P32这么便宜,才60-70?
行了 (这个不确定,说错了不要笑话我)其实买到的都是hot/cold ATP的混合物,放
时间长一些,就是hot/cold的ratio降低了
【在 l*******k 的大作中提到】 : 1 如果是gamma-32P ATP, 理论上还是可以被incoporate的,alpha-32P ATP应该就不行了 (这个不确定,说错了不要笑话我)其实买到的都是hot/cold ATP的混合物,放时间长一些,就是hot/cold的ratio降低了 : 2 放了一个月的还可以用,不过效率低了很多,最好用fresh的,我一般用14天之内的 : ,每个瓶子上都会标注时间 : 3 一瓶50微升的大概是六七十块钱吧,不是很贵
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l*******k 发帖数: 361 | 10 你要查瓶子上的标注,有一个specfic activity的,一般是3000或者6000,纯的hot
ATP 是 8000, cold ATP是0, 根据这个数据就可以算出hot/cold的ratio, specific
activity越高,说明Hot ATP 越多,这样得到的 probe 就越Hot,所以你买的时候记得买6000的而不是3000的
one more dumb question:
I heard from my colleague that they got "hotter"product by using a 10mCi
stock then a 250uCi stock. But that doesn't make much sense to me, as I
assume
one can only label one P32 to one RNA sequence, so if the kinase efficiency
is the same using either stock, the final product should be equally hot |
w******e 发帖数: 1187 | 11 thank you very much for the detailed reply!
specific
得买6000的而不是3000的
efficiency
【在 l*******k 的大作中提到】 : 你要查瓶子上的标注,有一个specfic activity的,一般是3000或者6000,纯的hot : ATP 是 8000, cold ATP是0, 根据这个数据就可以算出hot/cold的ratio, specific : activity越高,说明Hot ATP 越多,这样得到的 probe 就越Hot,所以你买的时候记得买6000的而不是3000的 : one more dumb question: : I heard from my colleague that they got "hotter"product by using a 10mCi : stock then a 250uCi stock. But that doesn't make much sense to me, as I : assume : one can only label one P32 to one RNA sequence, so if the kinase efficiency : is the same using either stock, the final product should be equally hot
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m*****z 发帖数: 1451 | 12 我用过2个月的,相当于不用加冷的,一样work。
好处是开罐的时候被照的少一点。
坏处是浪费钱。。。
【在 o**i 的大作中提到】 : p32 半衰14天 2个28天 : 我们lab从来不用7天以上的
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