Question

Below is a set of experiments for cloning the human growth hormone (HGH) gene and then expressing the HGH protein in E. coli starting from human pituitary glands and a tube of plasmid vector DNA. The plasmid expression vector contains the arabinose inducible expression system. Specify the correct order for carrying out these experiments, using the letter codes in front of each procedure. Use all of the steps in your answer, except for one step that should NOT be included.

A. Prepare liquid cultures of E. coli carrying the correct plasmid and add arabinose to the culture

B. Isolate mRNA from human pituitary glands

C. Analyze the plasmid clones by carrying out restriction digests followed by an agarose gel

D. Carry out PCR using primers designed to amplify the HGH coding sequence while simultaneously adding an EcoRI restriction site to one end and a XhoI site to the other end of the amplified fragment

E. Transform the reaction into E. coli

F. Isolate genomic DNA from human pituitary glands

G. Use T4 DNA ligase to ligate the purified gene of interest into a plasmid vector that has

been digested with EcoRI and XhoI

H. Carry out DNA mini-preps on several E coli colonies

I. Run the digested fragment on an agarose gel, and then purify the fragment from the gel

J. Use reverse transcriptase to synthesize cDNA from the mRNA

K. Digest the PCR-amplified fragment with EcoRI and XhoI

L. Run samples on SDS-PAGE

M. Verify the correct clone by DNA sequencing so that you don’t waste time and resources on doing the subsequent steps

N. Carry out western blotting to check for expression of the HGH protein

Answer 1

B. Isolate mRNA from human pituitary glands

J. Use reverse transcriptase to synthesize cDNA from the mRNA

D. Carry out PCR using primers designed to amplify the HGH coding sequence while simultaneously adding an EcoRI restriction site to one end and a XhoI site to the other end of the amplified fragment

K. Digest the PCR-amplified fragment with EcoRI and XhoI

I. Run the digested fragment on an agarose gel, and then purify the fragment from the gel

H. Carry out DNA mini-preps on several E coli colonies

G. Use T4 DNA ligase to ligate the purified gene of interest into a plasmid vector that has

been digested with EcoRI and XhoI

E. Transform the reaction into E. coli

C. Analyze the plasmid clones by carrying out restriction digests followed by an agarose gel

M. Verify the correct clone by DNA sequencing so that you don’t waste time and resources on doing the subsequent steps

A. Prepare liquid cultures of E. coli carrying the correct plasmid and add arabinose to the culture

L. Run samples on SDS-PAGE

N. Carry out western blotting to check for expression of the HGH protein

Thus,the order is BJDKIHGECMALN