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Studying genes at the Trancription& Translation level

Studying genes at the Trancription& Translation level - Molecular Cloning Forum

Studying genes at the Trancription& Translation level - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 10-28-2010, 04:54 PM
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Default Studying genes at the Trancription& Translation level



Hi,

I working on a strain that doesn't require a substrate to express the enzyme whereas other strian among the smae species require to. So, I am lil bit confused on how do I go about the whole thing?
Do I have to get the total RNA, do a real time PCR etc;??
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Old 10-28-2010, 06:05 PM
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Post Re: Studying genes at the Trancription& Translation level

Quote:
Originally Posted by Sr_08 View Post
Hi,

I working on a strain that doesn't require a substrate to express the enzyme whereas other strian among the smae species require to. So, I am lil bit confused on how do I go about the whole thing?
Do I have to get the total RNA, do a real time PCR etc;??
If you want to study trancripition and translation levels, that means you want the mRNA and protein levels.

You likely want a quantitative Real-time-RT-PCR (one-step rev-trancription-PCR is pretty nice) and maybe an ELISA or quantitative WB.

Another option for protein-of-interest quantitation, is either immuno-affinity or tag-specific purification, then a simple BCA protein assay of purified protein (with a SDS-gel for purity confirmation, gel quantitation w/ a standard and appropriate software is also possible).

Or if the enzyme activity can be measured, you can measure enzyme activity relative to amount of mRNA per ug sample/#cells (or whatever quantity).
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