Hi everyone, I'm working with unknown TCRs and I need to perform a 5' RACE PCR. Does anyone have a protocol to maximize the efficiency of the reaction?
I also would like to know if it's possible to do this technique without the use of a kit?
Thanks!!
I have cloned a gene without any kit and just took advantage of traditional 5' and 3' RACE techniques several years ago.
Firstly, buy an enzyme called TDT( terminal deoxynucleotidyl transferase ), dC and a poly-dG oligomer with proper adaptor; Then, add many dCs to purified cDNA with TDT and construction 5'-cDNA with poly-dG by PCR.
When doing 5'RACE PCR, you'd better do gradient PCR between 40 and 70 oC and do nested-pcr with low-fidelity taq enzyme.
Good luck!
I have cloned a gene without any kit and just took advantage of traditional 5' and 3' RACE techniques several years ago.
Firstly, buy an enzyme called TDT( terminal deoxynucleotidyl transferase ), dC and a poly-dG oligomer with proper adaptor; Then, add many dCs to purified cDNA with TDT and construction 5'-cDNA with poly-dG by PCR.
When doing 5'RACE PCR, you'd better do gradient PCR between 40 and 70 oC and do nested-pcr with low-fidelity taq enzyme.
Good luck!
I followed this protocol without buying the kit.
https://tools.thermofisher.com/content/sfs/manuals/5prime_race_man.pdf
Good luck
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