I want to do site directed mutagenesis to studying in vitro expression of natural mutants found by patients. So, I need the cDNA coding for the protein.
Is it difficult to prepare myself the cDNA? Someone has a protocol for me (products, methods, etc)?
If it is difficult, is it some manufacturers that could make it for me, and who?
Thanks a lot, I really need help
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1 reply to this topic
Posted 14 June 2002 - 09:12 AM
If the gene is known, then you can search in the litterature to get the sequences of the primers to be able to do a reverse transcriptase on RNA purified from the tissue you're interested in. Once you have the full-length RNA, you can proceed with PCR to get the cDNA. You then put this fragment into a vector of your choice (check the TA cloning kit to do PCR fragment that get easily in a vector). Once you have the vector containing the cDNA, you can do mutagenesis easily with commercial kit. This way, you can transfect the wild type and mutants into cells to see if the mutants lose their activity.