Question

You have cloned the Pepsi Gene into the vector pKEN. You used two restriction enzymes, BamHI and EcoRI to force the cloning in a directional way. The plasmid (pKEN) is ~5 kb long and the insert Pepsi 800bp long. After transformation into E. coli you grow up several colonies and isolate the vector. After digestion of the vector it looks like you have three potential clones. You need to sequence the gene to make sure you have the correct clone. However, you need to sequence both strands of the whole gene and the only primers you have generated are for the end of the Pepsi gene. Describe how you would sequence the whole gene including the ends of the gene.

Answer 1

The whole gene sequence can be identified by sequencing reactions. For sequencing reactions, we need sequencing primers at the ends of the gene that will give the sequence of around 800bp. Sanger Sequencing method can be used to get the sequence.