Hello,
I'm planning to perform an in vitro translation experiment to evaluate a coding potential of an RNA transcript (cca 1500 nt), predicted to code for a 8 kDa protein consisting of 68 aa residues. The problem that I run into is that I will need a positive control with the protein product of roughly the same molecular weight so that I'm able to tell that the translation system is working properly and whether my RNA transcript lacks coding potential.
Could anyone, please, suggest a protein of 8-10 kDa, encoded perhaps in a plasmid, which could be used as a positive control for this experiment?
I suppose describing the planned experiment could also be of help. The whole in vitro transcription/translation experiment will start with double digestion of a plasmid which contains promoter EF1a and a gene encoding for my RNA transcript. I plan to transcribe the gene from the fragment using HeLaScribe® Nuclear Extract in vitro Transcription System (Promega), modifying the RNA transcript on both ends (5' cap and 3' poly(A)) and performing the in vitro translation. I've looked into options regarding the in vitro translation kits and settled on Retic Lysate IVT Kit (Thermo Fisher) and radiolabelled L-[14C(U)]-Leucine (Perkin Elmer) since the predicted protein should contain 9 leucine residues (versus 3 methionine residues, which I understand to be a golden standard for radiolabeling). Any tips regarding these products or the overall assay will be more than welcome.
The kit control ( Specific transcription from the CMV Positive Control DNA results in a runoff transcript 363 nucleotides in length. ) not working?
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