Question

You wish to clone the human cnr1 gene into the DH5 alpha strain of E.coli. Design an outline for this project based on the PCR, Restriction digestion and complementation labs. Make sure you explain how you will acquire the gene sequence and the type of plasmid you plan on using.

Answer 1

I will design a primer for cnr1 gene with the NCBI sequence of the CNR1 gene that primer will have restriction sites and i will consider that the product should be in frame.

then i will perform the pcr amplification by using designed primer and cDNA as template( isolated from mammalian cells like HEK293).

Then i will perform PCR purification to remove the buffers and then i will load the amplified product into agarose gel to check the product followed by restriction digestion with corresponding enzyme. and at the same type I will digest the Vector like pGEX-6p2,pet28a with the enzyme followed by ligation and transformation and then insert release with particular enzyme.

pGEX-6p2,pet28a are bacterial expression plasmid if you want to clone to check the expression in mamallian exprssion you need to clone in pEGFP vector.