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cDNA cloning and Lentiviral transfection

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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cDNA cloning and Lentiviral transfection

Postby rkrishna » Thu Jun 27, 2013 11:22 pm

Hello,
I am new to molecular biology and have been facing some issues with my research.
My ultimate aim is to transfect mammalian cells so that they have integrin labelled with mCherry in them. I have this mcherry labelled integrin cDNA in a mammalian expression vector. If my understanding is correct, I can transfect cells with this vector directly to achieve my goal. But the amount of cDNA I have is very less, so I want to amplify it before transfection.
So my question is, Can I transform E.Coli with the vector with cDNA, and obtain copies of it? As in, can I use this vector for cloning in bacteria? Else, where do I start?
Also, I am considering lentiviral transduction of my mammalian cells as it has better efficiency. Is there a different method/ plasmids I need to use during cloning to be able to perform lentiviral transduction in later stages?
Any advice will be of help to me.
Thank you guys!
rkrishna
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Postby BlahBlahBlah » Fri Jul 12, 2013 1:09 pm

I would think that depends on the vector, which you've told us very little about....

The pWPT vector I used for lentiviral transfection has a bacterial promoter, origin of replication, and Ampicillin antibiotic resistance gene, to allow you to amplify it in bacteria.

I don't know what your vector is, so I can't say if you can or cannot (you probably can, most vectors are designed like that)
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