I would like to clone a cytokine in pet32 vector. The gene sequence was downloaded from NCBI and I checked the coding region for protein. How do I make sure that the gene is in alignment with the vector after cloning between two restriction sites?
I assume that you want to check that the ORF is conserved after cloning "in frame" with the Trx•Tag of the pET32. then you must sequence with a primer that anneals to the vector but close to the cloning site. Most commercial plasmids already have designed sequencing sites that anneal commercial primers, but you can make your own.
I agree that is the path one should follow before expression, but if one decides to go for a expression instead of sequencing. is there a way to ensure that you expression product is what you have cloned
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