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obtain a full length cDNA clone WITH RT-PCR


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#1 BILLO

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Posted 25 October 2010 - 07:14 AM

How to obtain a full length cDNA clone of thaumatin. which does not involve a library of cDNA clones. But based on RT-PCR. Also how much aminoacid sequence information might be available for the mature thaumatin protein to begin with (N term region vs full length of protein) and how can this information be used to design suitable thaumatin-specific primers to amplify ONLY thaumatin cDNA?
How would you ensure that ALL the pre-pro thaumatin coding sequence is copied the PCR.

#2 Curtis

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Posted 26 October 2010 - 12:29 AM

It is very simple; we have two types of RT-PCR, 1-step and 2-step methods. you probably want to do 2-step in which you make a cDNA with random hexamers/Oligo-dT and Reverse Transcriptase. Then you run PCR with Taq and the primers that you have designed with primer-BLAST software at NCBI website specific for that desired sequence. after that you run your PCR product on agarose gel to make sure it is the right size.

you better have a look on youtube or read a good textbook, but youtube better, saves time.

#3 BILLO

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Posted 26 October 2010 - 11:27 AM

It is very simple; we have two types of RT-PCR, 1-step and 2-step methods. you probably want to do 2-step in which you make a cDNA with random hexamers/Oligo-dT and Reverse Transcriptase. Then you run PCR with Taq and the primers that you have designed with primer-BLAST software at NCBI website specific for that desired sequence. after that you run your PCR product on agarose gel to make sure it is the right size.

you better have a look on youtube or read a good textbook, but youtube better, saves time.



Hey thanks for that....so if for example we have a purified protein and don`t know the amino acid sequence....and need to clone obtain full length cDNA clone of say `thaumatin` with using RT-PCR, how would we design the specific primers to amplify the thaumatin cDNA?

Thanks




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