transgene expression - (Jan/27/2007 )
This is my first post and I would like to collect some general information about overexpression of a gene.
I am about to start a new project in my lab. We are planning to overexpress a membrane protein in suspension culture tobacco cells. (I am not experienced in this field) The gene is from Arabidopsis and it is well charecerised and all the information about the gene is available in database.
So I am planning to isolate total mRNA from arabidopsis and get the cDNA of the corresponing gene and I would clone to a binary vector , trsnsformation, expression so and so......
Kindly advice me what are the crucial things which I have to take care during my project? Which binary vector would be the best choice? etc etc. I know its a boring qustion but all your suggestions will definitely make me confident. So please experts...help me with ur valuable advises.
Thank u very much
My major advice is to read as much about each technique as you can. The more you learn about your techniques the better you understand how they work and the more successful your experiments will be. Perhaps write down a list of your techniques, and starting with the first technique, look up posts in the relevant forums on that technique and read about them. You can browse the posts on this website. The marked posts in each forum should be particularly useful. Searching the archives is also a brilliant resource - that's at the bottom of the search page - you can also search the current posts as well in the search area. The biblic "Molecular Cloning: A Laboratory Manual" by Sambrook, Fritsch and Maniatis, the bible of molecular biology is also well worth reading - they are fantastic. The New England Biolabs (NEB) catalog also seems to have some good information at the back of it.
With regard to answering your specific question, my area of expertise is cloning, so my major piece of advice would be to choose the restriction sites you wish to clone your fragment into wisely. Vector religation is a major issue with cloning and good choice of restriction sites greatly reduces vector re-ligation and thereby increases the proportion of vectors that contain insert.
Here are my tips on choosing RE sites (they are mostly aimed at reducing vector re-ligation):
-choose different restriction sites on the 5`and 3` end of your sequence
-choose sites that create sticky ends, not blunt ends (aimed at improving ligation efficiency, blunt ends are a lot more difficult to ligate)
-choose sticky ends that have 4 bp overhangs
-choose restriction sites that are non-complementary, i.e. sites that have different bases in their overhangs so that there is no possibility that the bases will anneal to each other and cause the vector to re-ligate.
The best combination of restiction sites is to use one site that creates a 5` overhang and one that creates a 3` overhang because these sites cannot anneal to one another. There may be a lot of foreign expressions there but google them and you'll learn what they are.
Good luck, Rob
Thank you very much Rob
Could any one of u please give me some information about Suitable binary vectors, I mean some references for different binary vectors comparison study......thank u