木木 asked in 科學生物學 · 1 decade ago

有關於在Transfection時所要用的Kit ~~很急~

請問在Transfection and Luciferase Assay時使用的Dual-LuciferaseTM assay kit,此Kit 的原理(一定要說到),和大略的操作歩驟可不可描述一下~~

請了解此kit的大哥,大姊為我解答~~謝謝

Update:

可以再請問下~在此kit為什麼要加入兩種不同的luciferase protein, renilla luciferase protein 和firefly luciferase protein

還有luciferase要翻譯成冷光酶還是螢光酶呢?那一種才是正確的呢?

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  • 1 decade ago
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    Sorry to answer in English. I understand your question as 1) how the transfection works and 2) how the luciferase assay is performed with this Dual-Luciferase assay kit.

    1) Transfection can be done by applying the mix of liposome and your DNA that could potentially be transcribed to an mRNA and translated into proteins, or mRNA that could potentially translated into protein using cellular machinery. These nucleic acids coded reporter genes named firefly luciferase gene and renilla luciferase gene, which of could be on the same molecule of nucleic acid or separate nucleic acid. If they stay on the same construct then the construct is called Dual luciferase construct. Dual luciferase construct is mostly used for IRES study. If they stay separated, co-transfect both constructs are commonly used for transfection efficiency control. These reporters are widely used for promoter study and translation study in the past several decades.

    2) After these protein is translated in cell, you can easily break the cell by using the lysis buffer provided by the kit and harvest the lysate containing the luciferase proteins. Lysate can be further purified or not to proceed the luciferase light unit detection. You add the LARII in to you lysate (lysate:LARII=1:5 (v/v)) and measure the firefly light unit. The LARII contains the substrates that can be processed by firefly luciferase protein but not renilla luciferase protein and emit the light. Next, you add the same volume of stop & glo as you added of LARII to stop the firefly luciferase reaction and activate the renilla luciferase reaction. This "stop & glo" is the top secret of this kit that promega does not even sell it separately. Then you can measure the renilla luciferase activity after the stop and glo is added.

    2006-03-05 07:39:30 補充:

    Good luck to you!

    2006-03-05 17:48:59 補充:

    1) 一個是用來作control, 一個是用來實驗的。Control可以是用來normalize 細胞的總量,或是normalize transfection 的效率。2) 冷光酶吧?!Please also check 柏森's website:http://www.blossombio.com.tw/Products/DualReporter...

    Source(s): personal experience; promega website, http://www.promega.com/tbs/tm040/tm040.html
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