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preparing a vector for transfection


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#1 bayoutech1

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Posted 22 March 2015 - 06:22 AM

I am trying to develop a flowchart to prepare my mammalian vector for transfection. This is my thought process so far:

- grow plasmid in a growth medium with a selectable marker (Ab resistant)

- extract and purify DNA

 

somewhere I am getting lost or confused and have the following questions

don't I need to confirm that the promoter, ORF, poly A and sites for ribosome assembly are present before I begin transfection? how would I do this?

do I need to cut the plasmid with a restriction digest and have it self-ligate?

 

thanks for your help



#2 5280

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Posted 22 March 2015 - 08:16 AM

I assume you are working with a commercial vector and you or the company cloned the ORF. If it is a commercial vector then you do not need to worry about anything other than the ORF. Firstly you want to grow up your plasmid under antibiotic selection as mentioned from a single colony then purify transfection quality DNA - endotoxin free DNA preferably with a final concentration of at least 1ug/ul. Once you have this DNA you can perform a diagnostic restriction digestion to confirm your plasmid. Run this digest on a gel and the resulting banding pattern will be specific to your plasmid. Make sure you also run undigested plasmid alongside for comparison. You can also sequence your ORF. However, if this is a commercial vector then they probably sequence verified everything and provided this information on the datasheet. Sequencing information is important to make sure there were no mutations created during the cloning process which can happen when PCR is used for cloning. In that case, I would just do a digest just confirm you isolated the correct plasmid. Otherwise, if you are uncertain about the ORF then you can easily sequence it depending the insert size. Once you verified everything, you can perform a transient transfection in a mammalian cell line with this purified plasmid (you do not have to do anything to the plasmid to transfect it into a cell), harvest protein at 72hrs and perform Western blot analysis for the protein you are overexpressing. Make sure you also transfect cells with an empty vector and mock transfected as a control. 

 

Lastly, Addgene has some useful resources about this topic. 



#3 bayoutech1

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Posted 22 March 2015 - 10:56 AM

thanks. this was very helpful!






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