delete cDNA stop codon for fusion with fluorescent tag

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arcas
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delete cDNA stop codon for fusion with fluorescent tag

Post by arcas » Fri Apr 13, 2012 4:49 am

Dear all,

I intend to build a cDNA library and I intend to isolate my own cDNA from the norma RT kit. I intend to clone this cDNA into my own vector with fluorescent tag (YFP). However, I would need to remove the stop codon in the cDNA right? In this case, the cDNA sequence is unknown since I prepared it myself, thus, I am unable to design primers to delete the stop codon. My goal is to fuse this cDNA with the YFP tag in the vector so they can be expressed together. May I know how do I achieve this? Thank alot.

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JackBean
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Post by JackBean » Fri Apr 13, 2012 8:02 am

you want to make a cDNA-YFP fusion library or clone only one of the genes into vector with YFP?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

arcas
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Post by arcas » Fri Apr 13, 2012 8:41 am

Hi,

I am trying to make a cDNA-YFP library.

arcas
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Post by arcas » Fri Apr 13, 2012 10:10 am

Hi,

Can I use the normal reverse transcriptase kit to obtain the cDNA instead of those cDNA construction kit which contains a specialized vector? Im going to clone it into the vector of my choice.

And we decided to change our strategy. We are going to cut the cDNA with EcoRI(example) and include a linker with BamHI site(example). This is because BamHI will be used to cut the vector. Then with the linker on, we will digest the cDNA again with the BamHI to obtain the BamHI site which can then anneal to the BamHI site in the vector. Is that feasible?

Sorry for any confusion. Im kinda new to cloning and understand how tough it can be.

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