You have been given a project to study the newly discovered aromatase-kinase H (AKH). 3 -41 kDa protein. A colleague has isolated a -1400-bp Xho I restriction fragment of human CDNA that molecular probes indicate contains the akh gene. Your assignment is to clone the gene to produce sufficient AKH for further study.
• Insert the restriction fragment containing the akh gene into the PGLO
• Induce coli bacterial cells to take up the recombinant PGLO plasmid.
• Verify the presence of the PGLO plasmid carrying the akh gene in the bacteria by RFLP & PCR
• Verify the expression of the akh gene in the bacteria by purifying the AKH protein from transformed bacterial cultures.
Experiment 1.
Splice the Xho I restriction fragment containing the akh gene into the PGLO plasmid. Cut 10 Hg of PGLO plasmid with 5-times the theoretical minimum amount of enzyme in a minimum reaction volume. After cutting the plasmid, add 10 μl of the akh restriction fragment Ligate with 7.5 U enzyme in a total ligation reaction volume of 50 μl. Label this tube "pGLO + akh"
What volume of PGLO do you pipette into the digest reaction?
[__________] μl